structural analysis of carbohydrates by mass spectrometry
TRANSCRIPT
Purdue UniversityPurdue e-Pubs
Open Access Dissertations Theses and Dissertations
Fall 2013
Structural Analysis of Carbohydrates by MassSpectrometryChiharu KondaPurdue University
Follow this and additional works at: https://docs.lib.purdue.edu/open_access_dissertations
Part of the Analytical Chemistry Commons
This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] foradditional information.
Recommended CitationKonda, Chiharu, "Structural Analysis of Carbohydrates by Mass Spectrometry" (2013). Open Access Dissertations. 141.https://docs.lib.purdue.edu/open_access_dissertations/141
Graduate School ETD Form 9 (Revised 12/07)
PURDUE UNIVERSITY GRADUATE SCHOOL
Thesis/Dissertation Acceptance
This is to certify that the thesis/dissertation prepared
By
Entitled
For the degree of
Is approved by the final examining committee:
Chair
To the best of my knowledge and as understood by the student in the Research Integrity and Copyright Disclaimer (Graduate School Form 20), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material.
Approved by Major Professor(s): ____________________________________
____________________________________
Approved by: Head of the Graduate Program Date
Chiharu Konda
Structural Analysis of Carbohydrates by Mass Spectrometry
Doctor of Philosophy
Yu Xia
Peter T. Kissinger
Nikolai R. Skrynnikov
Hilkka I. Kenttamaa
Yu Xia
Robert E. Wild 10/25/2013
i
STRUCTURAL ANALYSIS OF CARBOHYDRATES BY MASS SPECTROMETRY
A Dissertation
Submitted to the Faculty
of
Purdue University
by
Chiharu Konda
In Partial Fulfillment of the
Requirements for the Degree
of
Doctor of Philosophy
December 2013
Purdue University
West Lafayette, Indiana
m/zm/z
p m/z
m/z
m/zp p
m/zp
pp
p
p
m/z
m/z pp m/z
m/zp m/z
m/zp
m/z m/z p
m/zm/z p
pp
pp
m/z p
m/z pp
m/z
p
m/z
m/z
p
pp
ppp p
p
p
m/z
m/z
m/z
p pp p p
p p p
m/z
m/z pp m/z
m/z pp p
p p pp
m/zm/z
p p pp
m/znative
m/z
native p p p
p
p p
p p
p p
p
p
p p
pp p
p p
pp p p
p
p pp p
pp pp
m/z
p
m/z
1
CHAPTER 1 INTRODUCTION
1.1 Carbohydrates
Carbohydrates (also called sugars, oligosaccharides, glycans) are defined as
polyhydroxyaldehydes, polyhydroxyketones and their simple derivatives, or larger
compounds that can be hydrolyzed into such units. A monosaccharide is the smallest unit
of carbohydrates which cannot be hydrolyzed into a simpler form. Free monosaccharides
can exist in ring or open-ring forms as shown in Figure 1.1. Changes in the orientation of
hydroxyl groups around specific carbon atoms generate new sugar molecules. For
example, mannose which has hydroxyl group on C2 is sticking up instead of down for
glucose and mannose is the C2 epimer of glucose. There are 3 chiral centers (C2-C4) and
8 (= 23) possible structures for D-monosaccharides and 16 possible structures (4 chiral
centers, C2-C5) if L-monosaccharides are included. The ring form of a monosaccharide
generates a chiral anomeric center at C1 for aldo sugars or at C2 for keto sugars. There
are two types of anomeric configurations, α and β, depending on the hydroxyl group is
sticking down or up, respectively.
2
Figure 1.1 Ring and open-ring forms of D-glucose.
Any sugar that has aldehyde group or be able to form one in solution through
isomerism is called “reducing sugars” and can attach to another monosaccharide via the
hydroxyl group of anomeric center. A sugar which two monosaccharides connected
through the newly formed bond (“glycosidic bond”) is called a disaccharide. Due to the
enormous number of combinatory ways to construct an oligomer, even a small subunit of
carbohydrates, disaccharide, can possibly have more than 104 structural isomers
(estimated by Laine1 as shown in Figure 1.2). Unlike oligonucleotides and proteins
which are expressed in a linear fashion, carbohydrates can form branched structures. In
addition, the hydroxyl groups are subjected to various modifications (i.e. acetylamine,
sulfate, or sialic acid groups). This structural complexity imposes challenges to any
existing analytical methods for complete structural characterization. The heterogeneity
of many carbohydrate samples also requires separation tools such as high performance
liquid chromatography (HPLC) to be coupled with analysis methods including mass
spectrometry (MS) and nuclear magnetic resonance (NMR).
α-D-Glucose β-D-Glucose
1
2
3
45
6
12
3
45
61
2
3
4
5
6
Ring form Ring formOpen-ring form
3
Figure 1.2 The number of possible structural isomers as increasing degree of polymerization (considering only aldohexoses and linear structure).
1.2 Glycosylation
Glycosylation is one of the most frequent post-translational modifications and
more than 50% of known proteins as well as 80% of membrane proteins are estimated to
be modified with glycans.2,3 Proteins and lipids modified with glycans are called
glycoproteins and glycolipids, respectively. Those glycan modifications are widely
involved in intermolecular and intercellular binding events from fertility to immunity.2 In
eukaryotic cells, there are two major classes of glycans according to the nature of the
linkage regions to the proteins: N- and O-linked glycans. An N-linked glycan (N-glycan)
is covalently linked to an asparagine residue of a peptide chain where the peptide
sequence matches with Asn-X-Ser or Asn-X-Thr (X could be any amino acid except
proline). N-glycans share a common core structure (Man3GlcNAc2) and can be divided
into three classes: high-mannose type, complex type, and hybrid type (Figure 1.3a).4 An
O-linked glycan (O-glycan) is typically linked to serine or threonine residue via N-
acetylgalactosamine (GalNAc). Unlike N-glycans, O-glycans have a variety of different
1.E+00
1.E+02
1.E+04
1.E+06
1.E+08
1.E+10
1.E+12
0 1 2 3 4 5#
of is
omer
sDegree of polymerization
4
structural core classes. The most commonly occurring O-glycan structures are shown in
Figure 1.3b.4 Since the discovery of glycoproteins in bacteria and their pathogenicity
relates to the glycan structure, bacterial glycoproteins are attractive targets for therapeutic
intervention.5,6 However, prokaryotic glycans consist of unusual monosaccharide units
and their analysis is much more challenging than already complex eukaryotic glycans.5,7
The development of sensitive analytical method for structural characterization which
does not depend on the previous knowledge of biological glycan synthesis is highly
desirable.
Figure 1.3 Common core structures for (a) N- and (b) O-glycans.
Complex type Hybrid type High-mannose type Core 1 Core 2 Core 3 Core 4
Galactose (Gal)
Glucose (Glc)
Mannose (Man)
N-Acetylneuraminicacid (Neu5Ac)
N-Acetylgalactosamine(GalNAc)
N-Acetylglucosamine(GlcNAc)
(a) N-glycans
(b) O-glycans
5
1.3 Overview of Glycan Structural Analysis
In order to understand the biological function of glycans, full-level of structural
information is necessary. Full-level of structural characterization of a glycan include the
following five levels: the identity (stereochemistry and modifications) of each
monosaccharide unit, the anomeric configuration of the glycosidic bonds, linkage
positions, branching location, and the sequence of the individual monosaccharides in the
oligomer (Figure 1.4).
Figure 1.4 Structural information necessary to fully characterize glycans.
NMR is the most powerful technique for structural analysis of glycans. The advantage of
NMR over other methods is that it offers the full-level of structural information in a non-
destructive way.8,9 The main drawback of this technique is that it typically requires large
amount of samples (μg to mg quantities) to characterize unknowns.9,10 In many cases,
such as the analysis of N- and O-linked glycans from glycoproteins, there is rarely
enough material available. The preferred techniques which are capable of smaller
quantity of samples (pg to ng) are enzymatic analysis or lectin affinity chromatography
Identity
AnomericConfiguration
Linkage
SequenceBranching location
6
coupled with MS or HPLC. Enzymatic analysis of glycans uses exoglycosidases which
are known to cleave bonds that are specific to the type of linkage, anomeric
configuration, and stereochemistry of monosaccharide. Selection of exoglycosidase
largely relies on the knowledge of biological synthesis. Sialidase (cleave sialic acid
which has linkage of α2-3, -6, and -8), β-galactosidase (β1-4 galactose), α-fucosidase
(α1-2, -3, -4, -6 fucose), β-N-acetylhexosaminidase (β1-2, -3, -4, -6 GlcNAc), α-
mannosidase (α1-2, -3, -6 mannose) are some examples of exoglycosidases.11
Susceptibility to cleavage by different kinds of exoglycosidases is assessed by analysis of
the reaction products by MS or HPLC.12-14 This approach gives a lot of structural
information if prior knowledge about the glycan is available (e.g. the glycan is an N-
glycan).15,16 However, the variation of exoglycosidases which have been isolated so far is
very limited to N-glycans. Especially most of exoglycosidases cleave multiple linkages,
thus linkage information cannot be obtained for completely unknown molecule. Lectins
are carbohydrate-binding proteins which recognize specific sugar moieties.17 Based on
the relative affinity of glycans to different lectins, each glycan is eluted differently in
lectin affinity chromatography and their structures can be identified.18,19 A variety of
lectins has been discovered and a series of lectin columns as well as lectin array is
available for glycan analysis. These methods allow high-throughput analysis. However,
the detail glycan structure is difficult to obtain since lectins recognize whole sugar
moieties instead of a single sugar unit resolution.
7
1.4 Structural Analysis of Glycans by Mass Spectrometry
Mass spectrometry was first applied to carbohydrate analysis in 195820 with
electron impact (EI) ionization.21,22 EI is said to be “hard” ionization method since it
induces extensive fragmentation and the molecular ions are typically not observed. Since
EI is only suitable for volatile organic molecules, hydroxyl groups in non-volatile
carbohydrates had to be protected by permethylation,23 or peracetylation24 to increase the
volatility prior to analysis.25 Since then, many researches for structural analysis of
glycans using mass spectrometry have been reported with the improvement of analytical
techniques, mass spectrometric instrumentation, as well as development of “soft”
ionization methods such as fast atom bombardment (FAB),26 electrospray ionization
(ESI)27,28 and matrix-assisted laser desorption ionization (MALDI)29,30 which enable
native carbohydrates to be ionized with molecular ion information to be obtained.
1.4.1 Ionization
FAB has been introduced in 198126 and was the first ionization method used in
MS experiment of carbohydrates. A mechanism of FAB is similar to EI, instead of
impacting the sample by electrons, FAB uses beam of atoms and the sample has to be in
the liquid matrix.26 The intact non-volatile compounds were ionized by FAB, especially
acidic oligosaccharides (containing Neu5Ac or sulfate) in negative ion mode worked
relatively well. However, the ion signal was not strong enough and the size of the
molecular weight applicable was typically low.31 Currently, FAB has been replaced by
later developed “soft” ionization methods: ESI and MALDI.
8
ESI and MALDI are two common methods for the analysis of carbohydrates.
Both ionization techniques were introduced in the late 1980’s and enabled non-volatile
and thermally labile compounds to be ionized. In ESI, the sample solution is highly
charged and sprayed through a capillary into a strong electric field to form a fine mist of
charged droplets. The solvent evaporates and produces gas-phase ions or solvated ions. In
order to be efficiently ionized, analytes are necessary to stay on the surface of the
droplets. However, the hydrophilicity of carbohydrates limits their surface activity in ESI
droplets, and thus ionization efficiency of carbohydrates is significantly lower than those
of peptides and proteins.32,33 NanoESI,34 which uses much lower flow rates down to some
tens of nL/min as compared to 1 to 10 μL/min for regular ESI, produces smaller charged
droplet sizes and enhances the ionization efficiency for carbohydrates.35
MALDI uses a matrix which contains small organic molecules that have strong
absorption at the laser wavelength for ionization. Before the analysis, analytes are
dissolved in the matrix, which are subsequently dried to form crystals. Intense laser pulse
ablates the surface of the dried crystal in vacuum. This ablation excites and sublimates
the matrix molecule into gas phase.36 Although the exact mechanism of the MALDI
process is still under debate,37,38 the widely accepted theory for ion formation involves
proton transfer in the solid phase before desorption. Ions in the gas phase are then
accelerated by the electric field towards the mass analyzer.38 Both ESI and MALDI
ionization methods have advantages and disadvantages for carbohydrate analysis. The
advantage of MALDI is that the ionization efficiency for neutral carbohydrates is
relatively constant as the size of the molecule increases, in contrast to ESI, where the
ionization efficiency decreases with an increasing molecular weight.39 The limitation of
9
MALDI comes from extensive fragmentation due to the higher internal energies
deposited to the ions by laser ablation as compared to ESI.
1.4.2 Derivatization Techniques for Glycan Analysis
Derivatization of sugars, such as permethylation of hydroxyl groups23,40 and
reducing end modification,41,42 reduces hydrophilicity of the sugar molecules and
increases the ionization efficiency significantly.43,44 Permethylation is the most widely
used modification for glycan analysis in mass spectrometry, especially after the
introduction of the solid phase permethylation method developed by Novotny
group.40,45,46 Solid phase permethylation allows simple and efficient permethylation
especially for small quantity of samples. The aldehyde group at the reducing end of
glycans can also react with alkylamines (typically aromatic amines, which also function
as chromophore for UV detection upon separation). Two approaches (amination and
reductive amination) of reducing end derivatization are summarized in Figure 1.5. Schiff
base formation by amination produces open-ring and closed-ring structures in
equilibrium. It has been confirmed by NMR and other techniques that closed-ring
(glycosylamine) structure is prevalent in solution with aromatic substitutions.47,48 The
Schiff base can be further reduced by sodium borohydride to form reduced Schiff base
which can only exist in the open-ring structure. While reduced Schiff base is more stable
than a glycosylamine, glycosylamine structure has been reported with analytical
advantages such as better HPLC separation and enhancement of cross-ring cleavages by
CID.47,49 A number of amine groups have been used as reducing end derivatives
containing cationic and anionic charges such as p-aminophenyl ammonium chloride
10
(TMAPA)50 and 5-aminosalicylic acid.33 TMAPA was shown to increase sensitivity
5000-fold under ESI condition relative to the native form oligosaccharides.50 These
charge-containing derivatizations do not only offer improved ionization efficiency but
also show positive effects on the fragmentation pattern of CID.51 Reducing end
derivatization (including isotopic labeling, i.e. 18O) can also be used to simplify the
complex fragmentation spectrum from CID by introducing mass differences of product
ions derived from the reducing end vs. non-reducing end. 41,42
Figure 1.5 Reaction scheme for amination and reductive amination.
1.4.3 Nomenclature for the Gas-phase Fragmentation of Oligosaccharides
The nomenclature for oligosaccharide fragmentation commonly used in the mass
spectrometry field has been introduced by Domon and Costello as shown in Figure 1.6.52
Fragment ions that contain a non-reducing end are labeled with uppercase letters A, B,
H2N-R2(-H2O)
open-ring(Schiff base)
closed-ring(glycosylamine)
reduction
Non-Reduced (NR) formby amination
open-ring(reduced Schiff base)
Reduced (R) formby reductive amination
open-ring
closed-ring (hemiacetal)
Native Glycan Labeled Glycan
11
and C, and those contain the reducing-end of the oligosaccharide or the aglycon are
labeled with X, Y, and Z; subscripts indicate the location of cleavage within the
oligosaccharide ion. B and Y ions are cleaved at the non-reducing side of a glycosidic
oxygen and C and Z ions are cleaved at the reducing side of a glycosidic oxygen. B, C, Y,
and Z ions are classified as “glycosidic bond cleavages.” A and X ions resulted from
cleavages across the glycosidic ring are termed as “cross-ring cleavages.” They are
labeled with superscript of two broken ring bond numbers that are assigned as shown in
Figure 1.6.
Figure 1.6. Nomenclature for glycoconjugate product ions generated by tandem MS.52
1.4.4 Structural Analysis of Oligosaccharides by Mass Spectrometry
Mass spectrometry is a widely applied method in structural analysis of
carbohydrates, due to its high sensitivity and capability of providing detailed molecular
information.53 However, obtaining full-level of structural information (sugar identity,
anomeric configuration, linkage position, sequence, and branching location) has not been
Y2 Z2 Y1 Z1 Y0 Z0
B1 C1 B2 C2 B3 C30,2A1
1,5X0
Reducing endNon-reducing end 0
12
34 5
0
12
34 5
12
possible by using only MS. The molecular weight information of carbohydrates is readily
obtained from soft ionization methods such as ESI33,54 and MALDI.29,39,55 Tandem mass
spectrometry (MS/MS) based on collision-induced dissociation (CID) is heavily relied on
to obtain structural information for carbohydrates. Glycosidic bond cleavages and/or
cross-ring cleavages are typically observed from collisional activation. Glycosidic bonds
are labile and can be easily cleaved under low-energy collisions. These fragments are
useful for sequencing and identifying branching location. On the other hand, cross-ring
cleavages need relatively higher-energy to induce and these fragments are useful for
identifying linkage positions. Sequence and branching location can be readily obtained
from MS2 CID of permethylated33,56-59 or peracetylated59,60 oligosaccharides in the
positive ion mode and from native oligosaccharide in the negative ion mode.61
Assignment of linkage positions between disaccharides can be obtained based on the
product ions generated by MS2 CID in negative ion mode49,62-66 and positive ion mode
with metal cation adducts.60,67-73 Linkage determination by MS2 CID has been applied to
oligosaccharides. As the molecular gets larger, it is more difficult for linkage
determination due to possible ion suppression of the diagnostic product ions for structural
determination.63,74 Stereochemistry of monosaccharides and anomeric configuration are
the most difficult structural information to obtain by MS. Notable examples to obtain
stereo-structure information include tandem mass spectrometry (MS2) of transition metal
chelated75 and amino acid complexed monosaccharides.76
13
1.4.5 MS2 vs MSn
Tandem mass spectrometry (MS/MS) is the key technique for structural analysis
by MS.77-81 MS2, the simplest MS/MS, has been routinely used for sequencing and
identifying branching locations by a series of product ions from glycosidic bond
cleavages. In order to identify linkage positions unambiguously, obtaining all of the
possible product ions from cross-ring cleavages is the key. Besides CID, different
activation/dissociation methods have been investigated, including infrared multiphoton
dissociation (IRMPD),82 electron capture dissociation (ECD),83,84 electron-induced
dissociation (EID),85 electron detachment dissociation (EDD),86 and electron excited
dissociation (EED).87 These methods have been shown to increase cross-ring cleavages
and are complementary to CID. However, the application of the above methods has been
limited to research groups which have these instrument capabilities. Overall, MS2 has
advantages of good sensitivity (pmol to fmol of sample quantiteis),88 high throughput,
and simplicity in performance and instrumentation. Depending on the nature of analytes
and the dissociation method used, key diagnostic ions (i.e. A type ions) may be missing
in MS2, leading to either miss-assigned or un-assigned linkage positions or other
structural information.63,74,89 Higher stages of tandem mass spectrometry (MSn, where
n>2), enables a sequential disassembly of the intact molecule, which can be back-tracked
based on the precursor-product relationships at each step. In general, MSn leads to higher
confidence in structural characterization, and is extremely useful in probing subtle
structure changes between isomers.57,90-92 Viseux et al. demonstrated the use of MS3 to
MS5 CID for structural characterization (sequence, linkage, and branching determination)
of linear and branched oligosaccharides. In their work, an unknown oligosaccharide was
14
first fragmented into disaccharide and/or trisaccharide substructures via CID. These
substructures were further subjected to CID and the fragmentation spectra were compared
to that from known reference molecules for linkage determination. By using the
substructures generated in MSn CID as opposed to the whole oligosaccharide, it
significantly reduced the size of reference molecules needed for comparisons and it
overcame the low mass cut off issue for detecting key ions with the use of
electrodynamic ion trap mass spectrometer.58 Furthermore, determining the identity of
low mass ions through comparison to a much more limited number of possible structural
isomers provides greater confidence in their structural determination, particularly in
discriminating between their unique stereochemical or regiochemical isomers.
1.4.6 A New MSn Approach for High Level Structural Analysis of Oligosaccharides
One of the grand challenges of oligosaccharide analysis is to develop a complete
approach based on MS toward a full level structural analysis of linear oligosaccharides by
obtaining information of sugar unit identity, anomeric configuration, linkage types and
sequence. We propose a new MSn approach to achieve this goal by 4 steps: (1) find a
diagnostic ion which gives any of the structural information listed above in the product
ions from MS3 CID of disaccharide ions (shown in the blue square in Scheme 1.1), (2)
make a standard spectral library by collecting MSn (n = 2 or 3) CID of the diagnostic ions
from all of the possible isomeric structures which was either synthesized or fragmented in
gas-phase from disaccharide ions, (3) find the optimized pathway to fragment down
oligosaccharide ions into overlapping disaccharide substructures (shown in the red square
in Scheme 1.1), and (4) employ spectral matching algorithm for easy and fair comparison.
15
Scheme 1.1 The MSn (n = 4 or 5) approach for detailed structural analysis of a linear oligosaccharide. Charges are not indicated on the structure. “M” stands for reducing end modification.
Figure 1.7 MS3 CID steps for sugar unit identity and anomeric configuration determination.93
HO 3 2 1O O M
3OOO4 2 1O O MMS1
MS3
MS3 MS4 MS3
MS4 MS4MS5
Diagnostic Ion
Disaccharide Substructure FormationHO
Disac3O OHOO4
cture Fcture F2O OHHO
SubstrO3 HO
n2 1O M
Y3 Y2
MS2
MS2MS2
C2
Anomeric Config.Identity Location Linkage
II. Disaccharide Ion Diagnostic Ion Structural Information
I. Oligosaccharide Ion Disaccharide Substructure Ions
-H -Sugar-GA (m/z 221)
Diagnostic Ion
MS3 CID
131
101113 22187 161
203
131
101113 22187 161
203
Sugar Identity Anomericity
Unknown Disaccharide-H -
MS2 CID
16
Finding the diagnostic ion which is a substructure of disaccharide ion is the key
concept of this approach since the number of structural isomers can be minimized and
significantly reduce the cost of library construction. Previous study by our collaborator,
Bendiak group, has demonstrated that the CID patterns of m/z 221 product ions (C8H13O7,
non-reducing sugar glycosidically linked to a glycolaldehyde (sugar-GA)) from
disaccharide anions can be used to determine the non-reducing sugar identity and
anomeric configuration (Figure 1.7).93,94 Using this m/z 221 diagnostic ion successfully
reduces the size of synthetic standards from 11520 possible structures (disaccharides) to
24 (monosaccharides) as shown in Figure 1.2. By following this concept, further
discovery of diagnostic ion for linkage type is expected. Next step is the application of
this structural analysis method to oligosaccharides. Oligosaccharides need to be broken
down into overlapping disaccharide substructures. This can be done by MSn (n = 2 or 3)
via fragmenting the oligosaccharide ions into a ladder of Yn-1 to Y2 ions, then, cleaving
off disaccharide substructures (C2 ion) from non-reducing end of each of the Y ions. MSn
(n>2) is typically performed using ion trap (Paul trap, linear ion trap, and Fourier
transform ion cyclotron resonance (FTICR)) in a tandem-in-time fashion. In practice, the
number of stages (n) of MS/MS that can be executed on a certain instrument is limited by
the number of ions remained after each step of ion activation and isolation.95 The loss of
the low abundance fragment ion could be significant for ion traps due to the space charge
effect.96,97 Due to the above reason, a systematic study of fragmentation chemistry of
oligosaccharides and the way to enhance the formation of desired ions are important as
well to accomplish multiple stages of MSn (n = 4 or 5) experiment.
17
Previously, Bendiak group successfully demonstrated the stereo-structure analysis
by the m/z 221 diagnostic ion for 1-2, 1-4, and 1-6 linked disaccharides. However, 1-3
linked disaccharides produced very few m/z 221 ions using ion trap CID, making it
difficult to perform MS3 analysis on the stand-alone ion trap instrument. We investigated
the unimolecular dissociation chemistry of 1-3 linked disaccharide anions under different
CID conditions to understand fundamental gas-phase ion chemistry for the formation of
m/z 221 diagnostic ion in Chapter 2. In Chapter 3, the MSn approach is discussed to
generate m/z 221 within linear oligosaccharides to obtain the stereo-structure information
of each sugar unit. Furthermore, we explored and found the new diagnostic ion for
linkage determination which is smaller than a monosaccharide, Z1 ion, and this Z1 ion
was used to obtain linkage information in linear and branched oligosaccharides in
Chapter 4. Finally, a variety of reducing end modification groups and their significance
on the fragmentation chemistry of oligosaccharides were studied in Chapter 5 in order to
enhance selective bond cleavages, which is critical in improving the sensitivity of the
overall MSn analysis.
18
1.5 Conclusions
MS has been extensively used in glycan analysis both independently and coupled
with other analytical techniques. Due to its high sensitivity and simplicity, it is highly
expected that the full-level characterization can be obtained only using MS. The current
limitation of MS method is that linkage position cannot be obtained from
oligosaccharides unambiguously under most of circumstances, as well as obtaining
stereo-structure (stereochemistry and anomeric configuration) information. As opposed to
the trend in this field using MS2, we focused on the development of MSn approach which
overcomes the current weakness of MS method on glycan analysis, obtaining
unambiguous information of stereochemistry, anomeric configuration, and linkage
position from oligosaccharides.
19
1.6 References (1) Laine, R. A., Glycobiology 1994, 4, 759-767. (2) Perkel, J. M., Science 2011, 331, 95-97. (3) Apweiler, R.; Hermjakob, H.; Sharon, N., Biochim. Biophys. Acta 1999, 1, 4-8. (4) Varki, A.; Cummings, R. D.; Esko, J. D.; Freeze, H. H.; Stanley, P.; Bertozzi, C. R.; Hart, G. W.; Etzler, M. E., Essentials of glycobiology. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 2009. (5) Dube, D. H.; Champasa, K.; Wang, B., Chem. Commun. 2011, 47, 87-101. (6) Balonova, L.; Hernychova, L.; Bilkova, Z., Expert Rev. Proteomics 2009, 6, 75-85. (7) Maki, M.; Renkonen, R., Glycobiology 2004, 14, R1-R15. (8) Vliegenthart, J. F. G.; Dorland, L.; Halbeek, H. v., High-Resolution, 1H-Nuclear Magnetic Resonance Spectroscopy as a Tool in the Structural Analysis of Carbohydrates Related to Glycoproteins. In Advances in Carbohydrate Chemistry and Biochemistry, Tipson, R. S.; Derek, H., Eds. Academic Press: 1983; Vol. 41, pp 209-374. (9) Duus, J. Ø.; Gotfredsen, C. H.; Bock, K., Chem. Rev. 2000, 100, 4589-4614. (10) Allen, S.; Richardson, J. M.; Mehlert, A.; Ferguson, M. A. J., J. Biol. Chem. 2013, 288, 11093-11105. (11) Hoja-Łukowicz, D.; Link-Lenczowski, P.; Carpentieri, A.; Amoresano, A.; Pocheć, E.; Artemenko, K.; Bergquist, J.; Lityńska, A., Glycoconj J 2013, 30, 205-225. (12) Guile, G. R.; Rudd, P. M.; Wing, D. R.; Prime, S. B.; Dwek, R. A., Anal. Biochem. 1996, 233, 205-211. (13) Rudd, P. M.; Gulle, G. R.; Kuster, B.; Harvey, D. J.; Opdenakker, G.; Dwek, R. A., Nature 1997, 388, 205-207. (14) Edge, C. J.; Rademacher, T. W.; Wormald, M. R.; Parekh, R. B.; Butters, T. D.; Wing, D. R.; Dwek, R. A., Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 6338-6342.
20
(15) Mechref, Y.; Novotny, M. V., Anal. Chem. 1998, 70, 455-463. (16) Geyer, H.; Schmitt, S.; Wuhrer, M.; Geyer, R., Anal. Chem. 1998, 71, 476-482. (17) Sharon, N.; Lis, H., Science 1989, 246, 227-234. (18) Cummings, R. D., Methods Enzymol. 1994, 230, 66-86. (19) Endo, T., J. Chromatogr. A 1996, 720, 251-261. (20) Finan, P. A.; Reed, R. I.; Snedden, W., Chem. Ind. (London) 1958, 36, 1172. (21) Bleakney, W., Phys. Rev. 1929, 34, 157-160. (22) Nier, A. O., Rev. Sci. Instrum. 1947, 18, 398-411. (23) Ciucanu, I.; Kerek, F., Carbohydr. Res. 1984, 131, 209-217. (24) Bourne, E. J.; Stacey, M.; Tatlow, J. C.; Tedder, J. M., J. Chem. Soc. 1949, 2976-2979. (25) Finan, P. A.; Reed, R. I., Nature 1959, 184, 1866. (26) Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N., Nature 1981, 293, 270-275. (27) Meng, C. K.; Mann, M.; Fenn, J. B., Z. Phys. D. 1988, 10, 361-368. (28) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F., Mass Spectrom. Rev. 1990, 9, 37-70. (29) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F., Int. J. Mass Spectrom. 1987, 78, 53-68. (30) Karas, M.; Hillenkamp, F., Anal. Chem. 1988, 60, 2229-2231. (31) Egge, H.; Peter-Katalinić, J., Mass Spectrom. Rev. 1987, 6, 331-393.
21
(32) Burlingame, A. L.; Boyd, R. K.; Gaskell, S. J., Anal. Chem. 1994, 66, 634R-683R. (33) Reinhold, V. N.; Reinhold, B. B.; Costello, C. E., Anal. Chem. 1995, 67, 1772-1784. (34) Wilm, M. S.; Mann, M., Int. J. Mass Spectrom. Ion Processes 1994, 136, 167-180. (35) Bahr, U.; Pfenninger, A.; karas, M.; Stahl, B., Anal. Chem. 1997, 69, 4530-4535. (36) Dreisewerd, K., Chem. Rev. 2003, 103, 395-425. (37) Zenobi, R.; Knochenmuss, R., Mass Spectrom. Rev. 1998, 17, 337-366. (38) Knochenmuss, R.; Zenobi, R., Chem. Rev. 2003, 103, 441-452. (39) Harvey, D. J., Rapid Commun. Mass Spectrom. 1993, 7, 614-619. (40) Mechref, Y.; Kang, P.; Novotny, M. V., Methods in Mol. Biol. 2009, 534, 53-64. (41) Harvey, D. J., J. Am. Soc. Mass Spectrom. 2000, 11, 900-915. (42) Harvey, D. J., J. Chromatogr. B 2011, 879, 1196-1225. (43) Karas, M.; Bahr, U.; Dulcks, T., Fresenius J. Anal. Chem. 2000, 366, 669-676. (44) Dell, A., Methods Enzymol. 1990, 193, 647-660. (45) Kang, P.; Mechref, Y.; Klouckova, I.; Novotny, M. V., Rapid Commun. Mass Spectrom. 2005, 19, 3421-3428. (46) Kang, P.; Mechref, Y.; Novotny, M. V., Rapid Commun. Mass Spectrom. 2008, 22, 721-734. (47) Her, G. R.; Santikarn, S.; Reinhold, V. N.; Williams, J. C., 1987, 6, 129-139. (48) Nishikaze, T.; Kaneshiro, K.; Kawabata, S.; Tanaka, K., Anal. Chem. 2012, 84, 9453-9461. (49) Li, D. T.; Her, G. R., Anal. Biochem. 1993, 211, 250-257.
22
(50) Hansson, G. C.; Karlsson, H., Methods Molec. Biol. Humana Press: Totowa, 1993. (51) Chen, S.-T.; Her, G.-R., J. Am. Soc. Mass Spectrom. 2012, 23, 1408-1418. (52) Domon, B.; Costello, C. E., Glycoconjugate J. 1988, 5, 397-409. (53) Zaia, J., Mass Spectrom. Rev. 2004, 23, 161-227. (54) Fenn, J.; Mann, M.; Meng, C.; Wong, S.; Whitehouse, C., Science 1989, 246, 64-71. (55) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T.; Matsuo, T., Rapid Commun. Mass Spectrom. 1988, 2, 151-153. (56) Reinhold, V. N.; Sheeley, D. M., Anal. Biochem. 1998, 259, 28-33. (57) Sheeley, D. M.; Reinhold, V. N., Anal. Chem. 1998, 70, 3053-3059. (58) Viseux, N.; de Hoffmann, E.; Domon, B., Anal. Chem. 1998, 70, 4951-4959. (59) Perreault, H.; Costello, C. E., J. Mass Spectrom. 1999, 34, 184-197. (60) Domon, B.; Müller, D. R.; Richter, W. J., Biol. Mass Spectrom. 1990, 19, 390-392. (61) Chai, W.; Lawson, A.; Piskarev, V., J. Am. Soc. Mass Spectrom. 2002, 13, 670-679. (62) Ballistreri, A.; Montaudo, G.; Garozzo, D.; Giuffrida, M.; Impallomeni, G.; Daolio, S., Rapid Commun. Mass Spectrom. 1989, 3, 302-304. (63) Garozzo, D.; Giuffrida, M.; Impallomeni, G.; Ballistreri, A.; Montaudo, G., Anal. Chem. 1990, 62, 279-286. (64) Lamb, D. J.; Wang, H. M.; Mallis, L. M.; Linhardt, R. J., J. Am. Soc. Mass Spectrom. 1992, 3, 797-803. (65) Carroll, J. A.; Willard, D.; Lebrilla, C. B., Anal. Chim. Acta 1995, 307, 431-447. (66) Mulroney, B.; Traeger, J. C.; Stone, B. A., J. Mass Spectrom. 1995, 30, 1277-1283.
23
(67) Domon, B.; Müller, D. R.; Richter, W. J., Org. Mass Spectrom. 1989, 24, 357-359. (68) Domon, B.; Muller, D. R.; Richter, W. J., 1990, 100, 301-311. (69) Zhou, Z.; Ogden, S.; Leary, J. A., J. Org. Chem. 1990, 55, 5444-5446. (70) Hofmeister, G. E.; Zhou, Z.; Leary, J. A., J. Am. Chem. Soc. 1991, 113, 5964-5970. (71) Lemoine, J.; Fournet, B.; Despeyroux, D.; Jennings, K. R.; Rosenberg, R.; de Hoffmann, E., J. Am. Soc. Mass Spectrom. 1993, 4, 197-203. (72) König, S.; Leary, J., J. Am. Soc. Mass Spectrom. 1998, 9, 1125-1134. (73) Xue, J.; Song, L.; Khaja, S. D.; Locke, R. D.; West, C. M.; Laine, R. A.; Matta, K. L., Rapid Commun. Mass Spectrom. 2004, 18, 1947-1955. (74) Dallinga, J. W.; Heerma, W., Biol. Mass Spectrom. 1991, 20, 215-231. (75) Gaucher, S. P.; Leary, J. A., Anal. Chem. 1998, 70, 3009-3014. (76) Augusti, D. V.; Carazza, F.; Augusti, R.; Tao, W. A.; Cooks, R. G., Anal. Chem. 2002, 74, 3458-3462. (77) Cooks, R. G.; Beynon, J. H.; Caprioli, R. M., Metastable Ions. Elsevier: Amsterdam, 1973. (78) Burinsky, D. J.; Cooks, R. G.; Chess, E. K.; Gross, M. L., Anal. Chem. 1982, 54, 295-299. (79) McLafferty, F. W., Tandem Mass Spectrometry. John Wiley: New York, 1983. (80) Busch, K. L.; Glish, G. L.; McLuckey, S. A., Mass Spectrometry / Mass Spectrometry: Techniques and Applications in Tandem Mass Spectrometry. VCH: New York, 1988. (81) Burinsky, D. J.; Cooks, R. G.; Chess, E. K.; Gross, M. L., Anal. Chem. 1982, 54, 295-299. (82) Xie, Y.; Lebrilla, C. B., Anal. Chem. 2003, 75, 1590-1598.
24
(83) Adamson, J. T.; Håkansson, K., Anal. Chem. 2007, 79, 2901-2910.
(84) Zhao, C.; Xie, B.; Chan, S.-Y.; Costello, C.; O’Connor, P., J. Am. Soc. Mass Spectrom. 2008, 19, 138-150.
(85) Wolff, J.; Laremore, T.; Aslam, H.; Linhardt, R.; Amster, I. J., J. Am. Soc. Mass Spectrom. 2008, 19, 1449-1458.
(86) Wolff, J.; Amster, I. J.; Chi, L.; Linhardt, R., J. Am. Soc. Mass Spectrom. 2007, 18, 234-244.
(87) Yu, X.; Huang, Y.; Lin, C.; Costello, C. E., Anal. Chem. 2012, 84, 7487-7494.
(88) Geyer, H.; Geyer, R., Biochim. Biophys. Acta 2006, 1764, 1853-1869.
(89) Dongre, A. R.; Wysocki, V. H., Org. Mass Spectrom. 1994, 29, 700-702.
(90) Li, B.; An, H.; Hedrick, J.; Lebrilla, C., Methods Mol. Biol. 2009, 534, 133-145.
(91) Ashline, D. J.; Lapadula, A. J.; Liu, Y.-H.; Lin, M.; Grace, M.; Pramanik, B.; Reinhold, V. N., Anal. Chem. 2007, 79, 3830-3842.
(92) Weiskopf, A. S.; Vouros, P.; Harvey, D. J., Anal. Chem. 1998, 70, 4441-4447.
(93) Fang, T. T.; Bendiak, B., J. Am. Chem. Soc. 2007, 129, 9721-9736.
(94) Fang, T. T.; Zirrolli, J.; Bendiak, B., Carbohydr. Res. 2007, 342, 217-235.
(95) McLuckey, S. A.; Glish, G. L.; Vanberkel, G. J., Int. J. Mass Spectrom., Ion Processes 1991, 106, 213-235.
(96) Cox, K. A.; Cleven, C. D.; Cooks, R. G., Int. J. Mass Spectrom., Ion Processes 1995, 144, 47-65.
(97) March, R. E., J. Mass Spectrom. 1997, 32, 351-369.
25
CHAPTER 2 DIFFERENTIATION OF THE STEREOCHEMISTRY AND ANOMERIC CONFIGURATION FOR 1-3 LINKED DISACCHARIDES
2.1 Introduction
A tandem mass spectrometry approach has been developed to differentiate the
stereochemistry and anomeric configuration for the non-reducing unit of hexose-
containing disaccharides having any of the 16 possible stereochemical variants.1,2 In this
method, diagnostic ions at m/z 221 were formed from CID of deprotonated disaccharide
ions (m/z 341). It was established that the m/z 221 ions consisted of the intact non-
reducing sugar glycosidically linked to glycolaldehyde, as indicated in Scheme 2.1
(where GA abbreviates glycolaldehyde). Note that an open-chain form for the reducing
sugar is indicated in Scheme 2.1 and also for other disaccharides discussed later. This is
based on the observation of absorbance in the carbonyl stretch region in variable
wavelength infrared radiation photo-dissociation of deprotonated monosaccharide anions
in the gas phase.3 When m/z 221 ions were further dissociated by collisional activation,
disaccharides having different non-reducing sugar units and anomeric configurations
showed distinct fragmentation patterns that matched synthetic glycosyl-GAs. This
method was shown to be useful for assigning the stereochemistry as well as the anomeric
configuration of the glycosidic bond for the non-reducing sugar in disaccharides having
1-2, 1-4, and 1-6 linkages.1,2 However, due to the low abundance of m/z 221 ions
26
produced from 1-3 linked disaccharides, MS3 CID of m/z 221 ions could not be
performed, and it was unclear whether the fragmentation patterns could be used for
assigning either their stereochemistry or anomeric configuration.
Scheme 2.1 Formation of glycosyl-GA anions at m/z 221 from CID of deprotonated 1-4 linked disaccharides.
In this chapter, a series of 1-3 linked disaccharides were studied on a hybrid triple
quadrupole-linear ion trap mass spectrometer (QTRAP 4000). MS3 CID data of m/z 221
ions from the 1-3 linked disaccharides were obtained for the first time. The formation of
m/z 221 ions was examined using different collisional activation methods, i.e. beam-type
CID and on-resonance ion trap CID of the deprotonated disaccharides. 18O-labeling of the
reducing carbonyl oxygen in 1-3 linked disaccharides was used to enable mass-
discrimination of structural isomers of the (usually) m/z 221 ions. By choosing the proper
CID conditions, the diagnostic m/z 221 ions (the glycosyl-GAs) could be formed as the
dominant isomer. Their CID fragmentation patterns could be used to establish the
stereochemistry and anomeric configuration of the non-reducing sugar unit from 1-3
linked disaccharides.
-H --H -
α-D-Glcp-(1-4)-Glc, m/z 341 α-D-Glcp-GA, m/z 221
CID CID
27
2.2 Experimental
2.2.1 Materials
A list of 7 disaccharides (6 reducing sugars and 1 non-reducing sugars) is shown
in Table 2.1. All samples were purchased from commercial sources (indicated by the
superscripts in Table 2.1) and used without further purification. H218O was purchased
from Sigma-Aldrich, Inc. (St. Louis, MO). α- and β-D-monosaccharide-glycolaldehyde
standards, glucopyranosyl-glycolaldehydes (Glcp-GA), galactopyranosyl-
glycolaldehydes (Galp-GA), and mannopyranosyl-glycolaldehydes (Manp-GA) were
synthesized as previously described.1 Disaccharides and synthetic standards were
dissolved in methanol to a final concentration of 0.01 mg/mL and NH4OH was added to a
final concentration of 1 % immediately before use.
Table 2.1 List of disaccharides being studied.
Analytes were purchased from: aSigma-Aldrich, Inc. (St. Louis, MO, USA) and bCarbosynth, Ltd. (Berkshire, UK).
Type Linkage
Disaccharides
1-2 α-D-Glcp-D-Glca β-D-Glcp-D-Glca
1-3α-D-Glcp-D-Glca β-D-Glcp-D-Glcb
α-D-Glcp-D-Frua α-D-Galp-D-Galb
α-D-Manp-D-Manb
28
2.2.2 18O-labeling of Reducing Disaccharides
Carbonyl oxygen of the reducing sugar was 18O-labeled by dissolving 0.5 mg of a
disaccharide or oligosaccharide in 100 μL of H218O and heated up to 60 °C for 6 - 24 h.
The progress of the reaction was monitored by MS. Once the reaction completed; the
above solution was further diluted to 0.01 mg/mL (30 μM) with methanol. 1% (volume)
of triethylamine was added to the diluted solution right before MSn analysis.
2.2.3 Mass Spectrometry
All samples were analyzed in the negative-ion mode on a QTRAP 4000 mass
spectrometer (Applied Biosystems/Sciex, Toronto, Canada) equipped with a home-built
nanoelectrospray ionization (nanoESI) source. A schematic diagram of the instrument
ion optics is shown in the Scheme 2.2. Two types of low energy collisional activation
methods were accessible on this instrument, i.e. beam-type CID and ion trap CID. In
beam-type CID, the precursor ions (m/z 341 or 343) were isolated in Q1, accelerated in
the Q2 collision cell for collisional activation, and all products were analyzed in the Q3
linear ion trap. Collision energy (CE) was defined by the potential difference (absolute
value) between Q0 and Q2. In ion trap CID, the precursor ions were isolated in the Q3
linear ion trap via the RF/DC mode and a dipolar excitation was used for collisional
activation. In order to perform ion trap CID at different Mathieu q-parameters, an AC
(alternating current) generated from an external waveform generator (Agilent
Technologies, Santa Clara, CA, USA) was used for resonance excitation. Frequency and
the low mass cut-off were calculated by SxStability (Pan Galactic Scientific, Omemee,
Ontario, Canada). MS3 CID experiments were carried out by first performing beam-type
29
CID of precursor ions in Q2. The fragment ions of interest were isolated in Q3 and then
subjected to ion trap CID. Analyst 1.5 software was used for instrument control, data
acquisition, and processing. The typical parameters of the mass spectrometer used in this
study were set as follows: spray voltage, -1.1 to -1.5 kV; curtain gas, 10; declustering
potential, 50 V; beam-type CID collision energy (CE), 5 to 30 V; ion trap CID activation
energy (AF2), 5 to 60 (arbitrary units); scan rate, 1000 m/z/s; pressure in Q2, 6.9x10-3
Torr, and in Q3, 3.6x10-5 Torr. Ion injection time was controlled to keep a similar parent
ion intensity: typically 3x106 counts per second (cps) for MS2 CID experiments and
1x106 cps for MS3 CID experiments. Activation time was kept constant at 200 ms for all
ion trap CID experiments. Seven spectra were collected for CID of m/z 221 ions from
synthesized monosaccharide-GA standards (deprotonated molecules) and disaccharides
over a one year period. Standard deviations of peak heights were calculated for major
fragments such as m/z 87, 99, 101, 113, 129, 131, 159, 161, 203, and 221, which were
observed from all the standards and disaccharides studied here except β-D-Glcp-GA and
β-D-Glcp-(1-2)-D-Glc, which showed no peaks at m/z 99.
Scheme 2.2 A schematic diagram of the QTRAP 4000, a hybrid triple-quadrupole/linear ion trap mass spectrometer.
Q1Q0 Q2 Q3nanoESI
Dipolar AC
Beam-type CID
Ion trap CID
5 mTorr 2.5x10-5 Torr
30
2.3 Results and Discussion
2.3.1 CID of Deprotonated Disaccharide Ions from 1-3 Linked Disaccharides
Ion trap CID of deprotonated 1-3 linked disaccharides (m/z 341) typically
generates ions at m/z 221 in trace abundance on a Paul trap instrument, and isolation or
further CID of m/z 221 ions have not been achieved before.2,4-6 The 4000QTRAP mass
spectrometer used in this study has a unique triple quadrupole-linear ion trap
configuration, offering high sensitivity due to the large capacity of the linear ion trap, and
allowing either beam-type or ion trap collisional activation. In beam-type CID, the
precursor ions were isolated in Q1 and accelerated in Q2 for collisional activation, while
ion trap CID was conducted in Q3 with a dipolar excitation for collisional activation.
Since CID fragmentation patterns can be sensitive to the means of activation, the
formation of m/z 221 ions from five 1-3 linked disaccharides was investigated via both
beam-type and ion trap CID. Figure 2.1 compares the MS2 beam-type and ion trap CID
of deprotonated β-D-Glcp-(1-3)-D-Glc (m/z 341) using low energy CID conditions. A
relatively low CE (6 V) was used for beam-type CID; in ion trap CID, the AF2 for an AC
dipolar excitation was set to 25 (arbitrary units) for 200 ms. Under either activation
condition, the absolute intensities of m/z 221 ions (indicated by an arrow in Figure 2.1)
were very low and their relative intensities were less than 1% (normalized to the base
peak in the spectrum). This phenomenon was generally observed for all 1-3 linked
disaccharides studied herein. The insets in Figure 2.1 demonstrate the isolated m/z 221
ions (with a 2 m/z isolation window) from each set of dissociation conditions. For beam-
type CID, 1x106 cps of m/z 221 ions could be accumulated with an injection time of 1 s,
31
which was sufficient for performing the next stage of tandem mass spectrometry (MS3 in
this case) with reasonable ion statistics and sensitivity. Far lower abundance of the m/z
221 ions (4.6x104 cps) could be isolated from ion trap CID of m/z 341, even after
doubling the injection time to 2 s. As a result, it was not feasible to obtain MS3 CID for
m/z 221 ions generated from m/z 341 precursor ions initially isolated within the trap. In
experiments described below, beam-type CID was used to dissociate disaccharide
precursor anions in the Q2 collision cell thereby generating m/z 221 product ions in high
enough abundance to acquire their spectra in the linear trap reproducibly.
Figure 2.1 MS2 CID spectra in the negative ion mode obtained from deprotonated β-D-Glcp-(1-3)-D-Glc (m/z 341) under low energy dissociation conditions: (a) beam-type CID (CE = 6 V), and (b) ion trap CID (AF2 = 25). Insets in (a) and (b) show the isolation of m/z 221 ions generated from beam-type CID (injection time = 1 s) and ion trap CID (injection time = 2 s), respectively.
100 140 180 220 260 300 340m/z
0
100
Rel
ativ
e In
tens
ity, %
341
161179
113143
100 140 180 220 260 300 340m/z
0
100179
161
341
113143
(a)
(b)
1.05e6221
222
Rel
ativ
e In
tens
ity,%
4.6e4 221
60
60
Inte
nsity
, cps
Inte
nsity
, cps
32
2.3.2 CID of m/z 221 Ions Generated from 1-3 Linked Disaccharides
Previous studies have demonstrated that m/z 221 product ions formed from
collisional activation of disaccharide anions typically consist of an intact non-reducing
sugar with a 2-carbon aglycon derived from the reducing sugar.2 Three dominant
fragment peaks are commonly observed from CID of m/z 221: m/z 101, 131 and 161.
The relative intensities of these peaks together with some other fragment ions can be used
to establish the fragmentation patterns and to distinguish the stereochemistry and
anomeric configuration of the non-reducing sugar. Given that the CID patterns of m/z
221 ions will be used for structural identification, spectral reproducibility is an important
issue. Similar to the findings from a Paul trap instrument,1,2 we noticed that the number
of ions (m/z 221) in the linear ion trap and the energy input into an ion were among the
most important parameters affecting spectral reproducibility. To ensure reasonable ion
statistics and avoid adverse space charge effects, the intensity of the m/z 221 ions was
kept at 1x106 cps before MS3 CID. Based on previous studies, the CID energies were
tuned so that the ratio of remaining precursor ion to the most abundant product ion was
kept around 18 ± 3%.1 Figures 2.2, a, b, e, and f were the averaged spectra from seven
repetitions collected over a one year period and they were further used to make spectral
comparisons in later discussion. Error bars in the spectra indicate the standard deviation
of the peak intensity for 10 major fragment ions which were frequently observed for all
the disaccharides studied herein (m/z 87, 99, 101, 113, 129, 131, 159, 161, 203, and 221).
The standard deviations for these peaks were less than 5% in most cases, indicating high
reproducibility of the spectra from day to day by controlling the ion counts in the trap
before CID and the energy input to the ions. Since the CID patterns upon dissociation of
33
m/z 221 ions can differ to some extent from instrument to instrument7, CID spectra of the
synthetic monosaccharide-GA were collected as standards for comparisons. Figures 2.2,
a and b show the CID data of - and β-D-Glcp-GA, respectively. The abundant peaks at
m/z 101 and 131 in Figure 2.2a are a signature of a non-reducing glucose with an
anomeric configuration. Note that a distinct fragmentation pattern is observed for the β
configuration (Figure 2.2b), where m/z 131 and 161 ions are dominant. The same
collisional activation conditions were applied to m/z 221 ions derived from α-D-Glcp-(1-
3)-D-Glc and β-D-Glcp-(1-3)-D-Glc, anomeric isomers containing a non-reducing
glucose. It is obvious that the spectra from the two anomeric isomers (Figures 2.2, c and
d) were drastically different from their corresponding D-Glcp-GA standards, however,
were similar to each other. This indicates that the m/z 221 ions generated using low
collision energies from m/z 341 precursors have different structures from the D-Glcp-GA
standards, and that their CID patterns cannot be used to assign either the stereochemistry
or anomeric configurations of the ions. Note that beam-type CID was used to generate
the m/z 221 ions from disaccharides shown in Figure 2.2, a condition differing from
previous studies where ion trap CID had been used.1 This difference in activation could
have contributed to the formation of structural isomers observed for the m/z 221 product
ions. In order to test this hypothesis, m/z 221 ions of 1-2 linked disaccharides, α-D-Glcp-
(1-2)-D-Glc and β-D-Glcp-(1-2)-D-Glc, were formed using similar beam-type CID
conditions and further subjected to MS3 CID (Figures 2.2, e and f). Except for a larger
fluctuation in peak intensity for m/z 203, almost identical fragmentation patterns to the
standards were observed (compare Figure 2.2, a to e and b to f), strongly indicating that
the expected D-Glcp-GA structures were formed. We further investigated a wide variety
34
of disaccharides and found that the CID patterns of m/z 221 ions matched with their
corresponding monosaccharide-GA standards with the exception of 1-3 linked
disaccharides when low collision energy beam-type CID conditions were used to
dissociate the disaccharides.
Figure 2.2 MS2 ion trap CID spectra of m/z 221 ions derived from synthetic standards (a) α-D-Glcp-GA, AF2 = 25 and (b) β-D-Glcp-GA, AF2 = 18. MS3 CID spectra of m/z 221 ions derived from low energy beam-type CID of glucose-containing disaccharides: (c) α-D-Glcp-(1-3)-Glc, CE = 6 V for MS2 and AF2 = 25 for MS3, (d) β-D-Glcp-(1-3)-Glc, CE = 6 V for MS2 and AF2 = 25 for MS3, (e) α-D-Glcp-(1-2)-Glc, CE = 5 V for MS2 and AF2 = 27 for MS3, and (f) β-D-Glcp-(1-2)-Glc, CE = 5 V for MS2 and AF2 = 25 for MS3. The error bars in the spectra show the standard deviation of the peak intensity based on seven spectra collected over a 1 y period.
-H -α-D-Glcp-GA, m/z 221
(b)
-H -β-D-Glcp-GA, m/z 221
-
60 100 140 180 220m/z
161
203131113
221
-H
α-D-Glcp-(1-3)-Glc, m/z 341
(c)
β-D-Glcp-(1-3)-Glc, m/z 341-H -
m/z 221
-
-H -α-D-Glcp-(1-2)-Glc, m/z 341
-H
β-D-Glcp-(1-2)-Glc, m/z 341
m/z 221 m/z 221
m/z 221
0
100
Rel
ativ
e In
tens
ity, %
0 60 100 140 180 220m/z
100
Rel
ativ
e In
tens
ity, %
161
203131113
221
(d)
60 100 140 180 220m/z
0
100
Rel
ativ
e In
tens
ity, %
60 100 140 180 220m/z
0
100
Rel
ativ
e In
tens
ity, %
87
99101
113
129
131
159161203
221
(a)
87101 113
129
131
159
161
203221
60 100 140 180 220m/z
0
100
Rel
ativ
e In
tens
ity, %
87101113
129159
161
221
131
(f)
60 100 140 180 220m/z
0
100
Rel
ativ
e In
tens
ity, %
87
101
113
129
131
159161203
221
99
(e)
35
18O-labeling at the carbonyl position of the reducing sugar was used to mass-
discriminate the “sidedness” of dissociation events to either side of the glycosidic linkage
and thus the origins of the m/z 221 and/or potential 223 product ions. Figure 2.3
compares relatively low energy beam-type and ion trap CID of 18O-labeled deprotonated
α-D-Glcp-(1-3)-D-Glc, m/z 343. Similar fragments were observed for both conditions;
however, the ion abundance for m/z 283 (loss of 60 Da, C2H4O2) was much higher in
beam-type CID relative to ion trap CID. It is possible that this fragmentation channel
requires higher activation energy and is promoted, even in lower-energy beam-type CID,
since higher collision energies (several eV) may have been obtained as compared to ion
trap CID (hundreds of meV). The inset in Figure 2.3a shows data collected using a wide
isolation window (6 m/z units) around m/z 221 after CID of m/z 343 precursor ions. A
peak at m/z 223, due to incorporation of 18O, appeared with much higher abundance than
m/z 221 ions for both low-energy beam-type and ion trap CID. Note that if the expected
D-Glcp-GA structure were formed, it should consist of the intact reducing sugar unit with
a 2-carbon aglycon derived from the reducing sugar (C-2 and C-3 or C-3 and C-4). In
this case, 18O should not be incorporated into the product ion and it should still appear at
m/z 221. Therefore, the observation of abundant m/z 223 ions indicated that under
relatively low energy CID conditions, most of the m/z 221 ions formed from α-D-Glcp-
(1-3)-D-Glc do not have the D-Glcp-GA structure which is the structural isomer needed
to distinguish the stereochemistry and anomeric configuration of the non-reducing sugar.
Ions at m/z 221 and m/z 223 were further subjected to ion trap CID. Figure 2.4
compares the CID spectra of the isolated m/z 221 ions and the m/z 223 ions generated
from 18O-labeled α-D-Glcp-(1-3)-D-Glc and β-D-Glcp-(1-3)-D-Glc. The CID spectra of
36
the m/z 221 ions (Figures 2.4, a and c) from the two anomeric isomers are distinct from
each other and almost identical to those of the corresponding α and β-D-Glcp-GA
standards (Figures 2.4, a and b). The fragmentation patterns of m/z 223 (Figures 2.4, b
and d), however, were similar to each other yet were very different from the synthetic
glucosyl-GA standards.
Figure 2.3 MS2 spectra of 18O-labeled α-D-Glcp-(1-3)-D-Glc under (a) relatively low-energy beam-type CID (CE = 6 V), and (b) ion trap CID (AF2 = 25). Insets in (a) and (b) show isolation of m/z 221 and 223 ions generated from beam-type CID (injection time = 1 s) and ion trap CID (injection time = 2 s), respectively.
100 140 180 220 260 300 340m/z
0
100R
elat
ive
Inte
nsity
, %343
283163179
100 140 180 220 260 300 340m/z
0
100
Rel
ativ
e In
tens
ity, %
343
179163
(a)
(b)
0
2.9e5
Inte
nsity
, cps
223
221
0
4.0e4
Inte
nsity
, cps
223
221
60
60
37
The major fragments that resulted from CID of the m/z 223 ions included product ions at
m/z 205, 163, 131, and 113. These ions are likely due to losses of water (-18 Da), a 2-
carbon piece, C2H4O2 (-60 Da), a 3-carbon piece including 18O, C3H6O2 + 18O (-92 Da),
and sequential or concerted losses of a 3-carbon piece plus water including 18O (-110
Da), respectively. Interestingly, neither the loss of water nor the loss of 60 Da
significantly involves loss of the 18O oxygen. The m/z 223 ions are hypothesized to have
a structure in which the reducing sugar is connected to a 2-carbon piece from the non-
reducing sugar as shown in the scheme above Figures 2.4b and 2.4d. Note that C-1 is no
longer chiral on the piece from the (former) non-reducing sugar, which also explains the
similarity in the CID data of the m/z 223 ion derived from the two anomeric isomers
(Figure 2.4, b and d). We also noticed some subtle differences between Figure 2.4b and
2.4d. For example, the relative intensities of m/z 205 and m/z 159 are higher (more than
10%) in Figure 2.4b than in 2.4d. These differences may be due to the existence of a
small fraction of structural isomers other than that hypothesized for the m/z 223 ions.
38
Figure 2.4 MS3 spectra of m/z 221 and 223 ions derived from 18O-labeled α-D-Glcp-(1-3)-D-Glc and β-D-Glcp-(1-3)-D-Glc. (a) CID of m/z 221 ions, CE = 15 V (MS2), AF2 = 15 (MS3), (b) CID of m/z 223 ions, CE = 5 V (MS2), AF2 = 14 (MS3) from 18O-labeled α-D-Glcp-(1-3)-D-Glc, and (c) CID of m/z 221 ions, CE = 15V (MS2) , AF2 = 28 (MS3), (d) CID of m/z 223 ions, CE = 5 V (MS2), AF2 = 24 (MS3) from 18O-labeled β-D-Glcp-(1-3)-D-Glc.
2.3.3 The Effect of CID Conditions on the Formation of m/z 221 Diagnostic Ions
As demonstrated in Figure 2.4, abundant structural isomers of m/z 221 product
ions, (m/z 223 ions from the 18O-labeled disaccharides) were observed under relatively
low-energy dissociation conditions of 1-3 linked disaccharides, either using beam-type or
ion trap CID. This prevents the assignment of the stereochemistry or anomeric
configuration of the non-reducing sugar in a typical scenario where either a disaccharide
(d)
-H - m/z 223 -H -
β-D-Glcp-(1-3)-Glc, m/z 343m/z 221 -H
-H --
β-D-Glcp-(1-3)-Glc, m/z 343
(c)
60 100 140 180 220m/z
0
100
Rel
ativ
e In
tens
ity, %
131
10199
22111387159
203
(b)(a)
-H - m/z 223 -H -
α-D-Glcp-(1-3)-Glc, m/z 343 m/z 221 -H
-H -
-α-D-Glcp-(1-3)-Glc, m/z 343
60 100 140 180 220m/z
163
205
113 131
223
0
100
Rel
ativ
e In
tens
ity, %
0
100
Rel
ativ
e In
tens
ity, %
159161
60 100 140 180 220m/z0
100
Rel
ativ
e In
tens
ity, %
163
113 205131
223159
60 100 140 180 220m/z
131
161
22187
203159101
1818
1818
39
is unlabeled or when it is isolated (unlabeled) from a larger oligosaccharide structure. It
would be highly desirable to optimize CID conditions or, for that matter, to find any
dissociation conditions whereby the relatively pure, structurally informative glycosyl-GA
(m/z 221) ions could be formed predominantly. Figure 2.5 shows the effect of collision
energies on the formation of m/z 221 and m/z 223 ions under beam-type and ion-trap
CID, using 18O-labeled β-D-Glcp-(1-3)-D-Glc as an example. The data were collected
using a wide isolation window around m/z 221 to observe both m/z 221 and m/z 223 ions.
It is clear from Figures 2.5a to c that the collision energy in beam-type CID
affects the absolute and relative intensities of m/z 221 ions. When the CE was relatively
low (CE = 5 V), m/z 223 ions were predominantly formed, with four times higher
intensity than that of m/z 221. At a higher CE (CE = 10 V), m/z 221 and m/z 223 ions
were seen at nearly equal intensities. Once the CE was increased to 15 V, m/z 221 ions
became the dominant peak, accounting for 80% of the total intensities from m/z 221 and
223. Further increasing CE, however, resulted in a huge loss of ion abundance possibly
due to competitive ion ejection thus the ratio was not improved.
Parameters that might affect the formation of m/z 221 ions versus m/z 223 ions
were also examined for ion trap CID. When ion trap CID of m/z 343 was performed
under the instrument default Mathieu q-parameter (q = 0.235), m/z 223 ions were formed
exclusively independent of activation energies (data not shown). By changing the
activation Mathieu q-parameter to a higher value, precursor ions are placed under a
higher potential well depth, and higher activation energies can be applied. An AC
generated from an external waveform generator was used for resonance excitation at q =
0.4. As shown in Figures 2.5d to f, the ratio of m/z 221 to m/z 223 ions was increased
40
from almost zero to about 1 as the activation amplitude was increased from 100 mVpp to
400 mVpp (activation time: 50 ms for all cases). Further increasing the activation
amplitude resulted in a decrease in m/z 221 to 223 ratio as well as a huge ion loss. The
data in Figure 2.5 suggest that m/z 221 ions, which have the desired monosaccharide-GA
structures, are generated more favorably under relatively high collision energy conditions
in both beam-type and ion trap CID. As compared to ion trap CID, beam-type CID
provided more abundant and higher relative intensities of the m/z 221 ions that were
wanted for discrimination of the stereochemistry and anomeric configuration of the non-
reducing sugar. Evidently, a higher activation energy is needed for the formation of these
m/z 221 product ions, and the pathway to generate the glycosyl-GAs is favored when the
internal energies of the molecular ions increase. In beam-type CID, much higher
collision energies can be applied (typically more than 10 V) as compared to ion trap CID
(hundreds of mV), which leads to a shift in the internal energy distribution of the
molecular ions to the high energy direction.8 It is interesting to point out that the
glycosyl-glycolaldehyde product ions are virtually the only isomeric species generated
under ion trap or low-energy beam-type CID of the 1-2 linked disaccharide anions.2
Since much higher relative intensities of the glycosyl-glycolaldehyde product ions (10 –
40%, normalized to the most abundant peak) can be formed from 1-2 linkages as
compared to that of 1-3 linkages (typically < 1% relative intensity) under ion trap CID
conditions, it is reasonable to conclude that the formation of these ions from 1-2 linkages
needs less energy than required for their generation from 1-3 linkages. Therefore, the
formation of the glycosyl-glycolaldehyde product ions is a much lower energy
dissociation channel for 1-2 linked disaccharides but a fragmentation pathway for this
41
isomeric species can only be promoted for 1-3 linked disaccharides when the collision
energy is higher.
Figure 2.5 Isolation of m/z 221 and m/z 223 ions derived from 18O-labeled β-D-Glcp-(1-3)-D-Glc under different collisional activation conditions. Beam-type CID: (a) CE = 5 V, (b) CE = 10 V, (c) CE = 15 V. Ion trap CID at q = 0.4, f = 119.248 kHz, excitation time = 50 ms: (d) 100 mVpp, (e) 250 mVpp, (f) 400 mVpp.
Given the high pressure in the collision cell (~ 5 mTorr), multiple collisions
happen in beam-type CID and the first-generation product ions may also be subjected to
collisional activation once they are formed within the collision cell especially under
higher CE conditions. In this sense, beam-type CID is less selective than ion trap CID,
where fragment ions are not typically further activated. Indeed, MS3 CID studies in the
ion trap showed that many fragment ions, including m/z 325, 323, 283, 281, 253, and 251
generated m/z 221 ions, which might contribute to the observation of higher intensity m/z
221 ions under beam-type CID due to secondary dissociation.
221 223m/z
3.9e5
Inte
nsity
, cps
223
221
221 223m/z
2.5e5221
223
221 223m/z
3.9e5 221
223
(a) (b) (c)
221 223m/z
9.1e4223
221 223m/z
2.0e5223
221
221 223m/z
1.0e5223
221(d) (e) (f)In
tens
ity, c
ps
Inte
nsity
, cps
Inte
nsity
, cps
Inte
nsity
, cps
Inte
nsity
, cps
0 0 0
0 0 0
42
2.3.4 Stereochemistry Determination of the Non-Reducing Sugar within 1-3 Linked Disaccharides
Since relatively pure m/z 221 ions containing the intact non-reducing sugars could
be formed using beam-type CID with high collision energies, it was possible to
differentiate the stereochemistry and anomeric configuration of the non-reducing sugar in
disaccharides without 18O-labeling. Figure 2.6 shows the MS3 CID spectra of m/z 221
ions generated by beam-type CID with relatively high CE (13 V to 22 V) from five 1-3
linked disaccharides. Each spectrum was an average of seven spectra and the error bars
indicate standard deviations of the peak intensities. The standard deviations were found
to be higher (0 - 12 %) for the disaccharide samples than those from standards (0 - 4 %).
This larger degree of spectral variation is likely contributed by the fluctuation in ion
intensity of the low abundance m/z 221 isomers under slightly different instrument
conditions, and these isomers fragment differently from the diagnostic and more
abundant m/z 221 ions that have the monosaccharide-GA structures. Note that α-D-Glcp-
(1-3)-D-Glc, α-D-Glcp-(1-3)-D-Fru, and β-D-Glcp-(1-3)-Glc are disaccharide isomers
containing a glucose as the non-reducing sugar; however, each has either a different
anomeric configuration or reducing sugar. The characteristic fragmentation profile for
disaccharides having glucose as the non-reducing sugar and an α-anomeric configuration
can be clearly identified for Figure 2.6a (α-D-Glcp-(1-3)-D-Glc) and Figure 2.6c (α-D-
Glcp-(1-3)-D-Fru), which is distinct from the β-anomeric isomer as shown in Figure 2.6b
(β-D-Glcp-(1-3)-D-Glc, compare all three to the synthetic standards shown in Figure 2.2a
and 2.2b). Figure 2.6d shows the characteristic fragmentation profile for the m/z 221 ion
43
of disaccharides having galactose as the non-reducing sugar and having an α-anomeric
configuration (compare to Figure 2.8a, CID of m/z 221 from -D-Galp-GA standard).
Figure 2.6 MS3 CID of m/z 221 ions generated via using high CE (CE = 13 to 22 V) for the dissociation of deprotonated disaccharide ions (m/z 341). (a) α-D-Glcp-(1-3)-D-Glc, CE = 15 V (MS2), AF2 = 26 (MS3), (b) β-D-Glcp-(1-3)-D-Glc, CE = 13 V (MS2), AF2 = 30 (MS3), (c) α-D-Glcp-(1-3)-D-Fru, CE = 18 V (MS2), AF2 = 25 (MS3), (d) α-D-Galp-(1-3)-D-Gal, CE = 22 V (MS2), AF2 = 35 (MS3), and (e) α-D-Manp-(1-3)-D-Man, CE = 20 V (MS2), AF2 = 36 (MS3). The error bars in the spectra show the standard deviation of peak intensities based on seven spectra collected over a 1 y period. The MS3 CID of α-D-Manp-(1-3)-Man (Figure 2.6e) was similar to that of the -D-
Manp-GA (Figure 2.8b). It is also important to note that under low-energy dissociation
conditions, the spectra of the m/z 221 product ions derived from the disaccharides α-D-
(b) (c)
-H -α-D-Glcp-(1-3)-Glc, m/z 341
m/z 221
β-D-Glcp-(1-3)-Glc, m/z 341-H - -H -
α-D-Glcp-(1-3)-Fru, m/z 341
-H -α-D-Galp-(1-3)-Gal, m/z 341-H -α-D-Manp-(1-3)-Man, m/z 341
m/z 221 m/z 221
m/z 221 m/z 221
(d)
60 100 140 180 220m/z
0
100
Rel
ativ
e In
tens
ity, %
87101113
129
131
159
161
203
221
60 100 140 180 220m/z
0
100
Rel
ativ
e In
tens
ity, %
87
99101
113
129
131
159161
203
221
60 100 140 180 220m/z
0
100
Rel
ativ
e In
tens
ity, %
8799
101
113129131
159
161
203221
60 100 140 180 220m/z
0
100
Rel
ativ
e In
tens
ity, %
8799101
113
129
131
159
161
203 221
(e)
60 100 140 180 220m/z
0
100
Rel
ativ
e In
tens
ity, %
8799
101
113
131
129 159
161
203221
(a)
44
Glcp-(1-3)-D-Fru, α-D-Galp-(1-3)-D-Gal and α-D-Manp-(1-3)-Man did not match those
of their respective glycosyl-glycolaldehydes (Figures 2.7 and 2.8c and 2.8d). We
conclude this is due to the presence of alternate isomers, possibly related in their origins
to the hypothetical structures shown in Figures 2.4b and 2.4d but having different
reducing monosaccharides.
Figure 2.7 CID spectra of m/z 221 ions derived from α-D-Glcp-(1-3)-D-Fru, CE = 5 V (MS2), AF2 = 30 (MS3).
60 100 140 180 220m/z
0
100
Rel
ativ
eIn
tens
ity, %
161
131
113
22120387
-H
α-D-Glcp-(1-3)-Fru, m/z 341
m/z 221
-
45
Figure 2.8 CID spectra of m/z 221 ions derived from (a) α-D-Galp-GA, AF2 = 17 (MS2), (b) α-D-Manp-GA, AF2 = 13 (MS2), (c) α-D-Gal-(1-3)-D-Gal, CE = 5 V (MS2), AF2 = 20 (MS3), (d) α-D-Man-(1-3)-D-Man, CE = 5 V (MS2), AF2 = 30 (MS3). The error bars in the spectra (a) and (b) show the standard deviation of the relative intensity calculated based on seven spectra collected over a 1 y period.
2.3.5 Spectral Matching by Similarity Scores
The methodology for assigning the stereochemistry and anomeric configuration
for the non-reducing sugar unit within a disaccharide is based on the comparison of the
CID patterns of m/z 221 ions to those of the synthetic monosaccharide-GA standards 1.
A high similarity between the compared spectra indicates a large likelihood of them
sharing the same structure. Spectral similarity scores, which have been widely used in
-H -α-D-Manp-GA, m/z 221-H -α-D-Galp-GA, m/z 221
-H - -H-
m/z 221
α-D-Galp-(1-3)-Gal, m/z 341 α-D-Manp-(1-3)-Man, m/z 341
m/z 221
(a)
100
60 100 140 180 220m/z
113131
161203
221101
(c)
0
Rel
ativ
eIn
tens
ity, %
60 100 140 180 220m/z
189161
131
203113
221
101
(d)
0
100
Rel
ativ
eIn
tens
ity, %
163
60 100 140 180 220m/z
0
100
Rel
ativ
e In
tens
ity, %
(b)
60 100 140 180 220m/z
0
100
Rel
ativ
e In
tens
ity, %
87 99 113129
131
159
161
203
221
8799
101
113131129
159
161
203 221
-
46
mass spectral library search for both small molecules,9 peptides10-12 and
oligosaccharides13 were chosen to facilitate these comparisons. The spectral similarity
scores were calculated between each of the averaged spectra in Figure 6 and the
averaged spectra from the monosaccharide-GA standards based on the following
equation,
Spectral similarity score , and k (Eq. 2.1)
where Im1 and Im
2 are the normalized intensities of an ion at m/z = m for the two spectra.
Note that the spectral similarity score always has a value between 0 and 1. If two spectra
are exactly the same, then, the spectral similarity score becomes 1. In general, a large
similarity score indicates close similarity between the two spectra and a large degree of
structural similarity. As shown in Table 2.2, the spectral similarity scores between a
standard and a disaccharide having the same stereochemistry and anomeric configuration
for the non-reducing side were the highest scores, ranging between 0.9838 and 0.9977.
When a disaccharide’s stereochemistry and anomeric configuration on the non-reducing
side did not match with the standard, the spectral similarity score was significantly lower,
between 0.6942 and 0.9178. Clearly, by comparing the spectral similarity scores,
assigning the stereochemistry and anomeric configuration for the non-reducing side of
the 1-3 linked disaccharides could be achieved with high confidence. Note that this was
only possible under high-energy beam-type CID conditions where the m/z 221 product
anions containing the intact non-reducing sugars were optimally generated from
precursor disaccharides.
47
Table 2.2 Spectral similarity scores for 1-3 linked disaccharides vs. monosaccharide-GA standards.
2.4 Conclusions
Collisional activation of deprotonated 1-3 linked hexose-containing disaccharides
(m/z 341) generated a low-abundance m/z 221 product ion. By 18O-labeling the reducing
sugar carbonyl oxygen of these disaccharides, at least two structural isomers of the m/z
221 ion with the main portion derived from either side of the glycosidic linkage could be
mass-discriminated (m/z 221 vs. m/z 223), which enabled the isomers to be isolated and
independently studied. The m/z 221 isomer containing the intact non-reducing sugar
attached in glycosidic linkage to a glycolaldehyde aglycon was found to be analytically
useful, since CID of this species provided the structural information that identified the
stereochemistry and anomeric configuration of the non-reducing sugar. No structural
information could be obtained from m/z 223 isomer(s) to determine the stereochemistry
of the non-reducing sugar or its anomeric configuration. The formation of the diagnostic
m/z 221 isomer was found to be affected by CID conditions and was favored under higher
energy beam-type CID. It was demonstrated that under optimized CID conditions, this
DisaccharidesSynthesized Standards
α-D-Glcp-GA β-D-Glcp-GA α-D-Galp-GA α-D-Manp-GAα-D-Glcp-(1-3)-Glc 0.9977 0.8845 0.8823 0.7968β-D-Glcp-(1-3)-Glc 0.8572 0.9840 0.7608 0.8568α-D-Glcp-(1-3)-Fru 0.9930 0.8899 0.9027 0.8045α-D-Galp-(1-3)-Gal 0.9178 0.7530 0.9838 0.6942α-D-Manp-(1-3)-Man 0.8461 0.8702 0.7527 0.9891
48
structural isomer could be generated predominantly from five different 1-3 linked
disaccharides without requiring 18O-labeling of the reducing sugar. Identification of the
nonreducing sugar and the anomeric configuration therefore were achieved at a high
confidence level by statistically comparing the CID data of m/z 221 ions generated from
the disaccharide samples to those of the synthetic standards via spectral similarity scores.
This study also demonstrated that beam-type CID was a more desirable activation method
as compared to ion trap CID to characterize disaccharides using the methodology based
on the CID patterns of m/z 221 ions. This method now enables the anomeric
configuration and stereochemistry of the m/z 221 ions derived from 2-, 3-, 4-, or 6-linked
disaccharides to be assigned in the negative ion mode. This capability was afforded due
to the specific arrangement of the triple quadrupole-linear ion trap instrument. It
combined (1) selection of the precursor (m/z 341) in the first quadrupole with (2) higher
energy dissociation in the second quadrupole collision cell followed by (3) buildup of the
desired low abundance m/z 221 product ion in the linear trap, all three of which were
necessary to obtain these structural details for 3-linked disaccharides.
49
2.5 References
(1) Fang, T. T.; Bendiak, B., J. Am. Chem. Soc. 2007, 129, 9721-9736.
(2) Fang, T. T.; Zirrolli, J.; Bendiak, B., Carbohydr. Res. 2007, 342, 217-235.
(3) Brown, D. J.; Stefan, S. E.; Berden, G.; Steill, J. D.; Oomens, J.; Eyler, J. R.; Bendiak, B., Carbohydr. Res. 2011, 346, 2469-2481.
(4) Dallinga, J. W.; Heerma, W., Biol. Mass Spectrom. 1991, 20, 215-231.
(5) Carroll, J. A.; Ngoka, L.; Beggs, C. G.; Lebrilla, C. B., Anal. Chem. 1993, 65, 1582-1587.
(6) Garozzo, D.; Giuffrida, M.; Impallomeni, G.; Ballistreri, A.; Montaudo, G., Anal.Chem. 1990, 62, 279-286.
(7) Bendiak, B.; Fang, T. T., Carbohydr. Res. 2010, 345, 2390-2400.
(8) Wells, J. M.; McLuckey, S. A., Collision-Induced Dissociation (CID) of Peptides and Proteins. In Biol. Mass Spectrom., Volume 402 ed.; Burlingame, A. L., Ed. Academic Press: 2005; pp 148-185.
(9) Stein, S.; Scott, D., J. Am. Soc. Mass Spectrom. 1994, 5, 859-866.
(10) Frewen, B. E.; Merrihew, G. E.; Wu, C. C.; Noble, W. S.; MacCoss, M. J., Anal. Chem 2006, 78, 5678-5684.
(11) Lam, H.; Deutsch, E. W.; Eddes, J. S.; Eng, J. K.; King, N.; Stein, S. E.; Aebersold, R., Proteomics 2007, 7, 655-667.
(12) Lam, H.; Deutsch, E. W.; Eddes, J. S.; Eng, J. K.; Stein, S. E.; Aebersold, R., Nat. Methods 2008, 5, 873-875.
(13) Kameyama, A.; Kikuchi, N.; Nakaya, S.; Ito, H.; Sato, T.; Shikanai, T.; Takahashi, Y.; Takahashi, K.; Narimatsu, H., Anal. Chem. 2005, 77, 4719-4725.
(14) Zhang, Z., Anal. Chem. 2004, 76, 3908-3922.
50
CHAPTER 3 OBTAINING STEREO-STRUCTURAL INFORMATION WITH SINGLE-SUGAR RESOLUTION FROM LINEAR OLIGOSACCHARIDES
3.1 Introduction
MS3 CID of the m/z 221 diagnostic ions was demonstrated to assign the
stereochemistry and anomeric configuration of the non-reducing sugar unit of D-
aldohexose (D-aldoHex) containing disaccharides for 1-2, -4, and -6 linked
disaccharides1 and 1-3 linked disaccharides2 in Chapter 2. This method was based on the
unique fragmentation patterns of diagnostic product ions formed by MS2 CID of
deprotonated disaccharide ions, having a non-reducing sugar glycosidically linked to a
glycolaldehyde (D-aldoHex-GA, m/z 221, C8H13O7; and D-aldoHexNAc-GA, m/z 262,
C10H17NO7 from acetylamine modified disaccharides). By comparing the MS3 CID
spectrum of thus obtained diagnostic ions from disaccharides to MS2 CID spectrum of the
synthetic standards using spectral matching algorithm, highly confident structural
assignment was achieved.2 Note that the diagnostic ion contains a substructure of a
disaccharide with significantly reduced number of stereo-centers, and thus the number of
standards for the diagnostic ion could be largely reduced (e.g. 16 for D-aldoHex-GA).
This is a significant reduction as compared to the fact that there are theoretically more
than 104 possible structural isomers for disaccharides (only considering D-hexoses).3
Twenty different sugar-GA standards have been synthesized so far (8 D-hexoses, D-
51
GlcNAc, and D-GalNAc, each with 2 anomeric configurations).1 These standards form
abundant diagnostic ions as the deprontated molecular ions ([M-H]-1) upon ESI in the
negative ion mode. MS2 CID of the synthetic standards provides a straightforward way
of generating standard spectra library on a given instrument. This approach has been
successfully applied for the determination of non-reducing sugars within 13 disaccharides
with a combination of 5 sugar units (D-Glc, D-Gal, D-Man, D-GlcpNAc, and D-
GalpNAc), 2 anomeric configurations, and 4 different linkage positions (1-2, -3, -4, and -
6).1,2
Based on the establishment of m/z 221 diagnostic ions for non-reducing sugar
identity and anomeric configuration from disaccharides, we attempted to develop an MSn
approach which can pinpoint individual sugar identity and anomeric configuration from
an oligosaccharide. The key characteristics of this MSn (n = 4, 5) approach involves
optimizing cleavages at glycosidic linkages during early stages of MSn to form
overlapping disaccharide substructures, then enhancing cross-ring cleavages for the
formation of the m/z 221 diagnostic ions, and finally obtaining the fragmentation pattern
of m/z 221 ions which can lead to the assignment of sugar unit identities and anomeric
configurations. A modified hybrid triple quadrupole/linear ion trap mass spectrometer
was employed in this study to enable high efficiency bidirectional ion transfer between
quadrupole arrays4 so that two types of CID, i.e., beam-type or on-resonance ion trap CID
can be executed at any stage of MSn up to 5 stages (MS5). This capability was shown to
be critical for optimizing the formation of the ladder of product ions involved in the MSn
analysis. This MSn approach using highly modified instrument successfully lead us to
garner detailed stereo-structural information of individual sugar units, i.e. sugar identity
52
and anomeric configuration, as well as their locations within two pentasaccharide
isomers: [α-D-Glcp-(1-4)]4-D-Glc and [β-D-Glcp-(1-4)]4-D-Glc.
3.2 Experimental
3.2.1 Materials
[α-D-Glcp-(1-4)]4-D-Glc, [β-D-Glcp-(1-4)]4-D-Glc, 4-aminobenzoic acid (ABA),
chloroform, sodium cyanoborohydride, acetic acid, and dimethyl sulfoxide (DMSO) were
purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Sugar-GA standards were
synthesized by Fischer glycosidation to generate allyl glycosides. They were converted to
the sugar-GAs by ozonolysis.1
3.2.2 Reducing End Modification (Reductive Amination)
Reducing end modification of oligosaccharides with ABA was conducted via
reductive amination.5 Briefly, oligosaccharides (0.5 mg) were dissolved in 10 μL of a
solution containing 0.15 M ABA in acetic acid: water (3:17, v/v), and 10 μL of 1.0 M
sodium cyanoborohydride dissolved in DMSO was added to achieve reductive amination.
After the vial was heated at 37 °C for 16 h, the solvent was removed under vacuum
(Speed-Vac, LABCONCO, Kansas City, MO) and the residue was dissolved in 300 μL
water followed by an addition of 300 μL chloroform. The aqueous phase containing the
derivatized oligosaccharide was collected after vortex mixing. This step was repeated
with another 300 μL of water for extraction and the aqueous phases were combined and
53
vacuum dried. The dried sample was dissolved in 100 μL DMSO and 900 μL methanol.
This solution was further diluted 100-fold in methanol prior to MS analysis.
3.2.3 Mass Spectrometry
A modified hybrid triple quadrupole/linear ion trap mass spectrometer (QTRAP
4000, AB SCIEX, Concord, Ontario, Canada) was used for performing experiments with
high efficiency bidirectional ion transfer between Q1, Q2, and Q3 quadrupole arrays.4
Ion selection in Q1 utilized RF/DC isolation, while ion isolation in Q2 and Q3 was
typically performed by broadband waveform at q = 0.45 for the center of the frequency
notch. Isolation of the m/z 221 diagnostic ion was performed with unit resolution before
the last stage of CID, while the isolation windows for other precursors were varied from
unit to 3 Th to achieve optimized sensitivity. Ion trap CID was performed in Q2 or Q3
through on-resonance excitation by applying a dipolar ac signal. Beam-type CID was
performed with energetic ion transfer either from the Q1 Q2 or from Q3 Q2.
Derivatized samples were diluted to 0.01 mg/mL in methanol with 1% ammonium
hydroxide for negative mode ESI, which was conducted at flow rates of 1-2 μL/min.
3.2.4 MS2 CID Database of 16 D-AldoHex-GA Synthetic Standards
MS2 ion trap CID of 16 deprotonated D-aldoHex-GA standards (m/z 221) was
collected in the Q3 linear ion trap on a QTRAP 4000 instrument. To avoid space charge
effects, the parent ion intensity before CID was kept around 1 x 106 counts per second
(cps). The remaining parent ion intensity after CID was kept around 18 ± 5% relative to
54
its base product ion (100%). Seven spectra from individual standards were collected over
a one year period. Standard deviations of peak heights were calculated for 21 major peaks
listed below, which were commonly observed from the standards. The averaged data
(from seven spectra) for the D-aldoHex-GA standards are shown in Figure 3.1, with the
standard deviations for each peak indicated. It is worth noting that depending on the
instrument and type of dissociation6 the specific product ion abundances for a single
compound can vary, but are reproducible within measured error bars for a specific
instrument, when energy input is defined by controlling the ratio of the precursor/base
product ion.
55
Figure 3.1 Averaged MS2 trap CID data of 14 deprotonated D-aldoHex-GA standards (m/z 221). The 21 major peaks were labelled with standard deviation based on 7 averaged spectra obtained over a 1 y period.
β-D-Galp-GA, m/z 221α-D-Galp-GA, m/z 221
β-D-Manp-GA, m/z 221α-D-Manp-GA, m/z 221
β-D-Altp-GA, m/z 221α-D-Altp-GA, m/z 221
8799
101
113 131129 159161
203 221
60 100 140 180 220m/z0
100
%87
131
129 203221
60 100 140 180 220m/z0
100
%
16122187
159
20310160 100 140 180 220m/z
0
100
%131
129 87 99
101
113129131 161 203 221
60 100 140 180 220m/z0
100
%
87101 129
131
161203 221
15960 100 140 180 220m/z
0
100
%
101
99131 159 221113 16185
60 100 140 180 220m/z0
100
%129
97
8799101
113129
131
159 161 203 221 87101113129
131
159
161
221
60 100 140 180 220m/z0
100
%
60 100 140 180 220m/z0
100
%
β-D-Glcp-GA, m/z 221α-D-Glcp-GA, m/z 221
α-D-Allp-GA, m/z 221
α-D-Talp-GA, m/z 221
β-D-Idop-GA, m/z 221α-D-Idop-GA, m/z 221
β-D-Allp-GA, m/z 221
β-D-Talp-GA, m/z 221
131
16122115911387 20397 141
60 100 140 180 220m/z0
100
%101
131203101
11387
161 221
60 100 140 180 220m/z0
100
%189
9785 129 141
101
113
2218985 20399 16112513160 100 140 180 220m/z
0
100
%
97131
201113
141129101161 221
203125
87 189159
60 100 140 180 220m/z0
100
%85
159
161
22112999 131113 20360 100 140 180 220m/z
0
100
%85
189
131113 22110187 20316160 100 140 180 220m/z
0
100
%85
β-D-Gulp-GA, m/z 221α-D-Gulp-GA, m/z 221161101
113203 221
143131 189
97177
60 100 140 180 220m/z0
100
%8985
131
159 221203161129 189
60 100 140 180 220m/z0
100
%8797101
56
3.2.5 Spectral Matching by Similarity Score
Spectral similarity scores were calculated between the CID spectrum of m/z 221
ions obtained from MSn (n =4 or 5) of oligosaccharides and each of the averaged spectra
in Figure 3.1 (CID spectra of m/z 221 ions of the D-aldoHex-GA standards). The
following equation was used for similarity score:7
Spectral similarity score , and k
where Im and Im* are the normalized intensities (to the highest fragment peak) of an ion at
m/z = m for the two spectra. 21 major peaks (m/z 85, 87, 89, 97, 99, 101, 111, 113, 125,
129, 131, 141, 143, 159, 161, 175, 177, 189, 201, 203, and 221) were chosen for this
calculation. Note that the spectral similarity score always has a value between 0 and 1. If
two spectra are exactly the same, then the spectral similarity score is equal to 1. In
general, a large similarity score indicates close similarity between the two spectra and a
large degree of structural similarity.
57
3.3 Results and Discussion
3.3.1 MSn Approach
The general strategy of the MSn approach is shown in Scheme 3.1. It involves
optimizing cleavages at glycosidic linkages during early stages of MSn, then enhancing
cross-ring cleavages only with disaccharides isolated as an ordered set of overlapping
substructures (illustrated with a tetrasaccharide in Scheme 3.1, where sugar unit number
was assigned from reducing end (i.e., unit 1 4). Key steps include: 1) dissociation of a
precursor derivatized at the reducing end (M, Scheme 3.1) to a ladder of smaller
oligosaccharides, each successively one sugar unit shorter, containing the reducing end
tag (Ym ions);8 2) generation of disaccharide fragments (C2 ions) from each Ym ion,
derived from the opposite end; 3) dissociation of the disaccharides to their m/z 221
diagnostic product ions (the sugar-GA); 4) fragmentation of these diagnostic ions; 5)
spectral matching of the diagnostic ion CID data to that of a database of synthetic
standards for assigning stereochemistry.
58
Scheme 3.1 The MSn (n = 4 or 5) approach for stereo-structural analysis of oligosaccharides. “M” stands for reducing end modification. Sugar unit number is assigned from the reducing end.
The above described MSn approach has the advantage of knowing the exact origin
of each fragment with respect to its initial position within the oligomer. Also, the
dissociation patterns of disaccharides in negative-ion mode provide information that can
assign the linkage between them.9 Given the need to preserve the structural integrity of
intact sugars and their linkage, disaccharides are key substructures to isolate during
earlier stages of MSn. The reducing end modification (M) introduces a mass distinction
between Y and C ions and also bears a negative charge that enables selection of the
ladder of singly charged Ym ions after MS2. Yet, through charge-transfer, it permits
59
disaccharide (C2) ions to be isolated via a neutral loss of the derivatized end of the
molecule (MS3). Note that this approach does not define the structure of the reducing end
unit.
The success of this method highly depends on producing reasonable yields of the
ladder of ions employed in MSn, i.e., Ym, C2, and m/z 221 ions, from a given
oligosaccharide. Formation of Ym and C2 ions requires glycosidic bond cleavages. These
bonds can be cleaved preferentially using low-energy CID under most conditions.10 Our
previous studies showed that the higher-energy beam-type CID condition was critical to
produce m/z 221 diagnostic ions with both high structural purity and reasonable yield for
many 3-linked disaccharides.2 On the other hand, ion trap CID of the m/z 221 ions was
necessary to obtain reproducibly distinct fingerprint patterns for structural identification.
In order to have the capacity of optimizing the product ions involved in the tree of MSn
analysis, it is desirable to perform experiments on an instrument platform which provides
both beam-type and ion trap CID at any MS/MS stage.
3.3.2 A Hybrid Triple Quadrupole/Linear Ion Trap for MSn
We choose to work on a hybrid triple quardrupole/linear ion trap instrument since
this type of instrument already has the capability of both beam-type and ion trap CID and
therefore, efforts of instrument modification can be significantly reduced based on this
platform. A schematic representation of the instrument is shown in Figure 3.2.
Conventionally, beam-type CID is performed by accelerating ions from Q0 to Q2 (Figure
3.2a), while the precursor ions are mass isolated in Q1 (RF/DC) during fly. In this mode,
beam-type CID can only be performed in the MS2 stage. However, if the flow of ions
60
can be reversed, the mass analysis quadrupole arrays (Q1 and Q3) and reaction sections
(Q0 and Q2) can be re-accessed and the tandem-in-space experiments such as beam-type
CID can be achieved as shown in Figure 3.2b and 3.2c. Indeed, Thompson et al.11 and
Xia et al.4 have shown that with the use of high efficiency bidirectional ion transfer
between quadrupole arrays, beam-type CID) can be performed in MSn (n>2) without
significant hardware modification of a Q-q-TOF instrument. Given the above
considerations, we employed a research grade triple quadrupole/linear ion trap mass
spectrometer to implement the proposed MSn approach. In order to facilitate high
efficiency bi-directional ion transfer, an axial electric field (LINAC)12,13 was superposed
to the center axis of Q2 and Q3 quadrupole arrays. The direction and amplitude of the
electric field were controlled from instrument software and were optimized during
experiments. Results collected from this instrument showed ~80% transfer efficiency
after three cycles of transfers (Q2 Q3, Q3 Q2, and Q2 Q3). Compared to beam-type
CID, it is relatively simple to perform ion trap CID at a given stage of MS/MS. The only
requirement is to transfer ions to the quadrupole array which is capable of ion trap CID.
On this modified instrument, on-resonance ion trap CID can be either performed in Q2 or
Q3 quadrupole arrays via dipolar ac excitation. Ion trap CID in Q2 improved CID
efficiency due to the higher pressure in Q2 (6 to 8 mTorr) as compared to that in Q3 and
Q1 (3 x 10-5 Torr), as previously described.14,15 With all above capabilities (i.e.,
reversing ion beams and ion trap CID in Q2 and Q3), MSn (n = 4 or 5) employing either
of the two CID methods is possible to conduct at any stage of MS/MS.
61
Figure 3.2 A schematic of a modified triple quadrupole/linear ion trap mass spectrometer (QTRAP 4000). Beam-type CID can be performed through energetic axial ion transfer between quadrupole arrays (Q0 to Q3): a) conventional method: Q1 to Q2 with mass isolation in Q1; b) reversed ion transfer from Q3 to Q2 with mass isolation in Q3; and c) reversed ion transfer from Q1 to Q0 with mass isolation in Q1.
3.3.3 Effect of CID Conditions on the Formation of Key Product Ions
A pentasaccharide modified with 4-aminobenzoic acid ([α-D-Glcp-(1-4)]4-D-Glc-
ABA, structure shown in the inset of Figure 3.3) was used as a model compound for
method and instrumental parameter optimization. Based on the MSn method described in
Scheme 3.1, an MS5 CID with following sequence is necessary to obtain sugar unit 3
structural information: [474 ([M-2H]2-) 624 (Ym) 341 (C2) 221 (diagnostic
ion) fragments]. Figure 3.3a shows the ion transfer scheme for conducting MS3 CID
using the modified triple quadrupole/linear ion trap. In short, the doubly charged parent
anions ([M-2H]2-, m/z 474) formed by ESI in negative ion mode, were mass selected in
the Q1 quadrupole array (RF/DC mode) and accelerated to the Q2 collision cell to effect
beam-type CID. Product ions were collected in Q3, followed by a broadband isolation of
Y3 (m/z 624) ions. Either beam-type or ion trap CID of m/z 624 ions could be performed
Q1Q0 Q2 Q3-’ve ESI
Auxiliary AC
3e-5 Torr 6-8 mTorr 3e-5 Torr10 mTorr
Beam CIDa)
Beam CID b)
Beam CIDc)
SK IQ1 IQ2 IQ3 EXST1 ST3
62
for MS3 by reversing the ion beam. Beam-type CID involved accelerating ions from Q3
to Q2 in the opposite direction of the traditional beam-type CID. For ion trap CID, m/z
624 ions were transferred from Q3 to Q2 with minimum kinetic energies, followed by on-
resonance dipolar ac excitation in Q2.
Note that the goal of the MS3 is to maximize the formation of C2 (m/z 341) from
Y3 (m/z 624), which requires a glycosidic bond cleavage between the first and the second
sugar units (refer to Figure 3.3 for the oligosaccharide nomenclature). A range of
collision energies (CEs) (10 – 60 V) were tested using beam-type CID from Q3 and Q2,
but m/z 341 ions were barely detectable under those conditions. At higher CEs, small
fragments in the range of m/z 200-300 dominated due to sequential fragmentation of Y3
ions (Figure 3.3b, CE = 42 V). The ion trap CID spectrum of Y3 ions (Figure 3.3c, q =
0.45, 1.4 V, and 300 ms activation) consisted of fewer fragments, most of which derived
from glycosidic bond cleavages including C2 ions (m/z 341) as well as m/z 462, 444, 300,
282. Ion trap CID consistently yielded more C2 ions from the set of singly charged Ym
ions as compared to beam-type CID. This observation is consistent with glycosidic bonds
being preferentially cleaved under slow heating conditions.16
Figure 3.3d shows the ion transfer scheme for conducting either beam-type CID
or ion trap CID in the 4th stage of MS/MS. The goal of MS4 is to maximize the formation
of m/z 221 diagnostic ions from C2 (m/z 341) ions. The MS1 and MS2 steps was the same
as Figure 3.3a and ion trap CID was employed in Q2 for the MS3 step. After that, the
MS3 product ions were sent to Q3 and C2 (m/z 341) ions were isolated in Q3 via
broadband. Using reversed ion transfer (Q3 Q2), C2 ions were subjected to either
beam-type or ion trap CID in Q2 for MS4. Our previous studies on disaccharides showed
63
that diagnostic product ions (m/z 221) were formed by cross-ring cleavages and usually
required higher activation energies, especially for 3- and 4-linked structures. Similarly,
C2 ions isolated from oligosaccharides required higher-energy CID to generate m/z 221
ions. As shown in Figure 3.3e, the m/z 221 diagnostic ion is the second most abundant
using beam-type CID (CE = 22 V), yet it is not detectable using ion trap CID (Figure
3.3f, q = 0.45, 1.4 V, and 300 ms activation). The data in Figure 3.3 demonstrate that the
capability of employing different types of CID is extremely important to optimize the
yields of key product ions, which is also critical to the success of the planned MSn
experiments.
64
Figure 3.3 MS3 and MS4 experiments of a pentasaccharide, [α-D-Glcp-(1-4)]4-D-Glc-ABA. (a) Ion transfer scheme for conducting either beam-type CID or ion trap CID in the 3rd stage MS/MS for the formation of C2 ions (m/z 341) via MS3 [474(2-) 624 fragments]). (b) MS3 beam-type CID from Q3 to Q2, CE = 42 V; (c) MS3 ion trap CID in Q2, q = 0.45, 1.4 V, and 300 ms of activation. (d) Ion transfer scheme for conducting beam-type or ion trap CID in the 4th stage MS/MS for the formation of m/z 221 diagnostic ions via MS4 [474(2-) 624 341 fragments] (e) MS4 beam-type CID from Q3 to Q2, CE = 22 V; (f) MS4 ion trap CID in Q2, q = 0.45, 1.4 V, and 300 ms of activation. The structure of [α-D-Glcp-(1-4)]4-D-Glc-ABA, is shown in the inset.
300
282222
624264246204 462365 444
0
100
%
m/z200 300 400 500 600 700
624
462444300282
341263 606179
c)
Beam CID
Trap CID
100 200 300 400
161
179341281
323143 263
m/z0
100 f)
Precursor ion Ion for further CID
Trap CID
221
Y3, 624C2, 341
342
1
5Unit #
b)
200 300 400 500 600 7000
100
%
m/z
341
%
MS3
MS3
MS4
161
221179143
263131 341100 200 300 400m/z
0
100e) Beam CID
%
MS4
[α-D-Glcp-(1-4)]4-D-Glc-ABA
Q1 Q2 Q3
Iso 474 Iso 624Beam CID
MSAE
c)Beam CIDd)Trap
CIDion transfer
MMM
))))))))c)BBBBBBBBeam) CCCCCICICICIDDDDdd)Trap)Trap
CICICICICICICICICICICCIC DDDDDDDDDDDDCIDCICICICICICICICIDDDDDDDD
Q1 Q2 Q3
Iso 474 Beam CID Iso 624
Iso 341TrapCID
MSAE
e)Beam CIDf)Trap
CIDion transfer
MMMMM
))))))))e)BBBBBBBBeam) CCCCCICICICIDDDDf)Trapf)T
CICICICICCICICICICICICICICIDDDDDDDDDDDDDDDCIDCICICICICCICICIDDDDDDDDp
a) MS3: [474 624 fragments]
d) MS4: [474 624 341 fragments]
65
3.3.4 Determination of Individual Sugar Identity and Anomeric Configuration from Pentasaccharides
The feasibility of determining individual sugar unit identity and anomeric
configuration within an oligosaccharide was tested with two model pentasaccharides: [α-
D-Glcp-(1-4)]4-D-Glc-ABA and [β-D-Glcp-(1-4)]4-D-Glc-ABA, isomers having - and
β-anomeric configurations, respectively. To determine the structural information for
sugar units from the non-reducing end (i.e. unit 5 2), the following ions need to be
produced at MS2: m/z 341 (C2), 786 (Y4), 624 (Y3), and 462 (Y2), respectively. Both
singly (1-) and doubly (2-) deprotonated parent ions were formed from ESI in the
negative mode. Beam-type CID (CE = 21 – 24 V) of 2- charge state parents yielded
higher intensities for most product ions thus beam-type CID was chosen for MS2 prior to
higher stages of MSn. Using sugar unit 3, for example, the set of MS5 experiments
required to obtain its stereo-structural information were: [474(2-
) 624 341 221 fragments]. Based on previous study, ion trap CID was used for
MS3 of m/z 624 ions and beam-type CID was used for MS4 CID of m/z 341. Ion transfer
scheme specific to this MS5 experiment is shown in Figure 3.4a and CID conditions of
each step were summarized in Table 3.1. MS5 CID of m/z 221 was obtained for both
pentasaccharide isomers differing only in their anomeric configurations. For [α-D-Glcp-
(1-4)]4-D-Glc-ABA, the m/z 221 fragment derived from sugar unit 3 (Figure 3.4b)
yielded a fragmentation pattern characteristic of α-D-Glcp-GA (Figure 3.4d, error bars
from 7 experiments acquired over a 1 yr period). Spectral matching to the D-aldoHex-GA
standard database (16 structures: 8 sugar identities × 2 anomeric configurations) was
based on similarity scores calculated as described in the experimental section. α-D-Glc
66
was identified as the top match for sugar unit 3 with a high similarity score (0.9771) as
compared to the second match (β-D-Ido, 0.9171, Table 3.1). Figure 3.4c shows the MS5
CID spectrum of [β-D-Glcp-(1-4)]4-D-Glc-ABA acquired from the same MSn sequence.
This spectrum is markedly different from the corresponding α-anomer (Figure 3.4b) and
almost identical to the ion trap CID data of synthetic β-D-Glcp-GA (Figure 3.4e). Here,
β-D-Glc was the top match (similarity score: 0.9654) in comparing to the database of
standards. The alternate anomers matched poorly (scores of 0.75-0.86, Table 3.1). The
data in Figure 3.4 demonstrate that even for a subtle structural difference, i.e. anomeric
configuration, sugar unit 3 could be confidently determined.
Figure 3.4 MS5 experiments of pentasaccharides, [α-D-Glcp-(1-4)]4-D-Glc-ABA and [β-D-Glcp-(1-4)]4-D-Glc-ABA: (a) Ion transfer scheme for conducting MS5 CID for both pentasaccharides. MS5 ion trap CID of (b) [α-D-Glcp-(1-4)]4-D-Glc-ABA and (c) [β-D-Glcp-(1-4)]4-D-Glc-ABA. MS2 ion trap CID of synthetic standards: (d) α-D-Glcp-GA and (e) β-D-Glcp-GA.
[α-D-Glcp-(1-4)]4-D-Glc-ABA [ -D-Glcp-(1-4)]4-D-Glc-ABA
0
100
%
100 150 200m/z
131
101
16111387 221159
(b) MS5
100 150 200m/z
131
161
87 159 221
(c)
0
100
%
MS5
(d)
100 150 200m/z
0
100
%87
99101
113129
131
159161203
221
MS2(e)
87101113129
131
159
161
221
100 150 200m/z
0
100
%
MS2
a) MS5:[474 624 341 221 frag]
Q1 Q2 Q3
Iso 474 Beam CID Iso 624
Iso 341TrapCID
MSAE
Beam CID
TrapCID
Iso 221
67
Determining sugar units 5 and 4 involved MS4 [474(2-) 341 221 fragments]
and MS5 [474(2-) 786 341 221 fragments] experiments. The data are shown in
Figures 3.5 and 3.6 for [α-D-Glcp-(1-4)]4-D-Glc-ABA and [β-D-Glcp-(1-4)]4-D-Glc-
ABA, respectively. Conditions for each MSn stage are summarized in Table 3.2. Sugar 2
could not be identified since m/z 221 ions were not formed by CID of the Y2 ion. Table
3.1 summarizes the top 3 similarity scores of diagnostic ions derived from the two
pentasaccharides against the D-aldoHex-GA standard database. A full list is reported in
Table 3.3. The highest scores were assigned to the correct identity and anomeric
configuration for each sugar unit. The stereo-structures of 3 individual sugar units were
successfully determined for each pentasaccharide based on MSn (n = 4 or 5) experiments
and spectral matching to standards.
68
Figure 3.5 MSn (n = 4 or 5) for the determination of stereo-structural information of sugar units 3-5 from [α-D-Glcp-(1-4)]4-D-Glc-ABA. Spectra (a) to (c) show the sequential MS2 to MS4 data based on [474(2-) 341 221 fragments] to determine sugar unit 5 structural information. Spectra (d) to (f) show the sequential MS3 to MS5 data based on [474(2-) 786 341 221 fragments] to determine sugar unit 4. Spectra (g) to (i) show the sequential MS3 to MS5 data based on [474(2-) 624 341 221 fragments] to determine sugar unit 3. The experimental conditions for each step can be found in Table 3.2.
221
161143 179
263
383
425 786444341
282 606 7680
100
%
200 300 400 500 600 700m/z
800 0
100
150 200 250 300 350m/z
131
101
113 159 2211611299987 141 2030
100
100 150 200m/z
0
100
150 200 250 300 350m/z
900
545
587474768503444341 7860
100
%
300 400 500 600 700 800m/z
161
179
221 263281341
338
131
159101 221113 16199
12987 141 2030
100
100 150 200m/z
[M-2H]2-
a) b) c)
d) e) f)
624
Precursor ionIon for further CID
[α-D-Glcp-(1-4)]4-D-Glc-ABA34 2 15Unit#
0
100
I%
m/z200 300 400 500 600 700
624
462444300282
341263 606179
161
221179143
263131 341100 200 300
m/z
0
100
0
100
100 150 200m/z
131
101
16111387 221159
g) h) i)
MS2 MS3 MS4
MS3 MS4 MS5
MS3 MS4 MS5
69
Figure 3.6 MSn (n = 4 or 5) for the determination of stereo-structural information of sugar units 3-5 from [β-D-Glcp-(1-4)]4-D-Glc-ABA. Spectra (a) to (c) show the sequential MS2 to MS4 data based on [474(2-) 341 221 fragments] to determine sugar unit 5 structural information. Spectra (d) to (f) show the sequential MS3 to MS5 data based on [474(2-) 393(2-) 341 221 fragments] to determine sugar unit 4. Spectra (g) to (i) show the sequential MS3 to MS5 data based on [474(2-) 624 341 221 fragments] to determine sugar unit 3. The experimental conditions for each step can be found in Table 3.2.
131
161
221
m/z
87 101100 150 200
m/z
282
474
503 786393341
[M-2H]2-
222545
587
200 500 800
161
221179
143341
m/z100 200 300 400
393
282
624341
m/z200 400 800600
161
221
179143
263 341
m/z100 200 300
131
161
221
87 101
m/z100 150 200
624
a) b) c)
d) e) f)
Precursor ionIon for further CID
[ -D-Glcp-(1-4)]4-D-Glc-ABA34 2 15Unit#
0
100
%
0
100
%
0
100
0
100
0
100
0
100
400
200 300 400 500 600m/z
462
444300
282
179263
624341222 606
100 200 300 400m/z
161
143 179 221113 263
100 150 200m/z
131
161
87 159 221730
100
%
0
100
0
100g) h) i)
MS2 MS3 MS4
MS3 MS4 MS5
MS3 MS4 MS5
70
Table 3.1 The top 3 ranked D-aldoHex candidates based on spectral similarity scores (indicated in parenthesis).
Like any MSn experiment, each stage of MS/MS enhances the selectivity of the
experiment at the cost of reduced ion signal. The signal loss arises from partitioning of
parents among fragments and ion loss during isolation/dissociation. Six orders of
magnitude reduction of signal was observed in MS5 experiments. This led to long signal
averaging periods to achieve good signal/noise (S/N) ratios. For instance, Figure 3.4b
accumulated 31160 scans (~11 hr) to reach a S/N of 50, using 6-7 μg of sample. The
number of scans, however, can be lowered using the spectral matching approach. For
example, fewer scans (3422, ~1.2 hr) than the spectrum shown in Figure 3.4b with a
lower S/N (7) still provides the first-ranked spectral similarity score (0.9475) against α-
D-Glcp-GA (second match: β-D-Idop-GA, 0.8928). Note that these spectra could not be
obtained at all using only one type of dissociation methods, for example; there was
simply not enough m/z 221 product ion to afford a spectrum without beam-type CID.
sample Unit # Spectral Similarity Score1st 2nd 3rd
[α-D-Glcp-(1-4)]4-D-Glc-ABA
3 α-D-Glc(0.9771)
β-D-Ido(0.9171)
β-D-Alt(0.9084)
4 α-D-Glc(0.9939)
β-D-Alt(0.9082)
β-D-Ido(0.9070)
5 α-D-Glc(0.9862)
β-D-Alt(0.8913)
β-D-Ido(0.8902)
[β-D-Glcp-(1-4)]4-D-Glc-ABA
3 β-D-Glc(0.9654)
α-D-Ido(0.9407)
α-D-Alt(0.9253)
4 β-D-Glc(0.9802)
α-D-Ido(0.9649)
α-D-Alt(0.9498)
5 β-D-Glc(0.9748)
α-D-Ido(0.9639)
α-D-Alt(0.9499)
1
Tabl
e 3.
2 E
xper
imen
tal c
ondi
tions
for c
ondu
ctin
g M
Sn exp
erim
ents
.
Supe
rscr
ipts
indi
cate
the
figur
es, a
bove
, fro
m w
hich
cor
resp
ondi
ng M
S da
ta w
as d
eriv
ed.
Sam
ple
Uni
t #M
S2M
S3M
S4M
S5
[α-D
-Glc
p-(1
-4)] 4
-D
-Glc
-AB
A
347
4(2-
)[S
2a]
Q1
Q2
Beam
(24
V)47
4(2-
)62
4[S
2g]
Q2
Trap
(q =
0.4
5, 1
.4 V
, 300
ms)
474(
2-)
624
341
[S2h
]
Q3
Q2
Beam
(24
V)47
4(2-
)62
434
122
1[S
2i, 4
a]
Q3
Trap
(q =
0.2
3, 1
5 m
V, 1
00 m
s)
447
4(2-
)Q
1Q
2 B
eam
(23
V)47
4(2-
)78
6[S
2d]
Q2
Trap
(q =
0.4
5, 1
.8 V
, 100
ms)
47
4(2-
)78
634
1[S
2e]
Q3
Q2
Beam
(24
V)47
4(2-
)78
634
122
1[S
2f]
Q3
Trap
(q =
0.2
3, 1
8 m
V, 1
00 m
s)
547
4(2-
)Q
1Q
2 B
eam
(23
V)47
4(2-
)34
1[S
2b]
Q3
Q2
Beam
(23
V)47
4(2-
)34
122
1[S
2c]
Q3
Trap
(q =
0.2
3, 1
8 m
V,1
00 m
s)
------
-----
------
-----
------
----
[β-D
-Glc
p-(1
-4)] 4
-D
-Glc
-AB
A
347
4(2-
)Q
1Q
2 Be
am (2
3 V)
474(
2-)
624
[S3g
]
Q2
Trap
(q =
0.4
5, 1
.4 V
, 300
ms)
474(
2-)
624
341
[S3h
]
Q3
Q2
Beam
(22
V)47
4(2-
)62
434
122
1[S
3i,4
b]
Q3
Trap
(q =
0.2
3, 1
4 m
V, 1
00 m
s)
447
4(2-
)Q
1Q
2 B
eam
(21
V)47
4(2-
)39
3(2-
)[S
3d]
Q3
Q2
Beam
(15
V)47
4(2-
)39
3(2-
)34
1[S
3e]
Q3
Q2
Beam
(16
V)47
4(2-
)39
3(2-
)34
122
1[S
3f]
Q3
Trap
(q =
0.2
3, 1
2 m
V, 1
00 m
s)
547
4(2-
)[S
3a]
Q1
Q2
Bea
m (2
2 V)
474(
2-)
341
[S3b
]
Q3
Q2
Beam
(23
V)47
4(2-
)34
122
1[S
3c]
Q3
Trap
(q =
0.2
3, 1
5 m
V, 1
00 m
s)---
-----
------
------
-----
------
-
71
2
Tabl
e 3.
3 S
pect
ral s
imila
rity
scor
es fo
r eac
h un
it in
olig
osac
char
ides
aga
inst
16
D-a
ldoH
ex-G
A st
anda
rds.
Th
e nu
mbe
rs in
red
indi
cate
the
high
est s
core
s (f
irst m
atch
) w
hile
the
num
bers
in b
lack
bol
d in
dica
te th
e se
cond
hig
hest
sco
res
(sec
ond
mat
ch).
Sam
ple
Uni
t #
Aldo
hexo
se-G
A Sy
nthe
ticSt
anda
rds
α-D
-All
β-D
-All
α-D
-Alt
β-D
-Alt
α-D
-Gal
β-D
-Gal
α-D
-Glc
β-D
-Glc
α-D
-Glu
β-D
-Glu
α-D
-Idoβ-
D-Id
oα-
D-M
anβ-
D-M
anα-
D-T
alβ-
D-T
al
[α-D
-Glc
p-(1
-4)] 4
-D
-Glc
-AB
A
30.
8094
0.76
730.
8656
0.90
840.
8994
0.78
570.
9771
0.85
560.
8543
0.85
720.
8599
0.91
710.
8140
0.86
310.
9008
0.78
38
40.
7940
0.75
440.
8863
0.90
820.
8844
0.81
930.
9939
0.86
290.
8006
0.86
510.
8723
0.90
700.
8137
0.85
210.
8567
0.74
11
50.
7701
0.76
850.
8862
0.89
130.
8517
0.83
120.
9862
0.85
970.
7785
0.88
360.
8867
0.89
020.
8361
0.82
070.
8406
0.71
83
[β-D
-Glc
p-(1
-4)] 4
-D
-Glc
-AB
A
30.
4633
0.73
890.
9253
0.59
290.
6621
0.81
450.
7962
0.96
540.
6712
0.82
730.
9407
0.69
910.
8466
0.53
120.
7047
0.59
96
40.
4708
0.72
860.
9498
0.60
320.
6647
0.86
360.
7910
0.98
020.
6544
0.83
910.
9649
0.67
100.
8520
0.56
270.
6690
0.60
40
50.
4099
0.70
750.
9499
0.54
270.
6094
0.87
210.
7559
0.97
480.
6092
0.85
270.
9639
0.64
760.
8507
0.51
320.
6443
0.57
28 72
73
3.4 Conclusions
An MSn approach to pinpoint the stereo-structures (sugar identity, anomeric
configuration, and location) of individual sugar units within linear oligosaccharides was
described. The approach involved first optimizing the isolation of disaccharide units as an
ordered set of overlapping substructures via glycosidic bond cleavages. Subsequently,
cross-ring cleavages were optimized for individual disaccharides to yield key diagnostic
product ions (m/z 221). High confidence stereo-structural determination was achieved by
matching MSn CID of the diagnostic ions to synthetic standards via a spectral similarity
scores. By using this approach, structural information (identity and amoneric
configuration) of 3 individual sugar units (out of 5) was successfully determined for two
isomeric pentasaccharides. To our knowledge, this is the first report showing that specific
sugar identities, their anomeric configurations, and their positions within
oligosaccharides can be determined by MS. The above described approach of higher
stages of MSn could be used as a complementary means to NMR for oligosaccharide
structural analysis while offering enhanced sensitivity. A larger pool of oligosaccharides
will be tested for the applicability in future studies. In the case of branched
oligosaccharides (N-glycans), exoglycosidases will be used to convert the branched
structure into linear structures before subjected mass spectrometric analysis. We will also
explore different types of reducing end modifications which could enhance fragmentation
at selected bonds within the oligosaccharides. Several instrumentation methods are being
evaluated for enriching minor fragment ions at any MSn stage for improved sensitivity.
74
3.5 References
(1) Fang, T. T.; Bendiak, B., J. Am. Chem. Soc. 2007, 129, 9721-9736.
(2) Konda, C.; Bendiak, B.; Xia, Y., J. Am. Soc. Mass Spectrom. 2012, 23, 347-358.
(3) Laine, R. A., Glycobiology 1994, 4, 759-767.
(4) Xia, Y.; Thomson, B. A.; McLuckey, S. A., Anal. Chem. 2007, 79, 8199-8206.
(5) Harvey, D. J., J. Am. Soc. Mass Spectrom. 2000, 11, 900-915.
(6) Bendiak, B.; Fang, T. T., Carbohydr. Res. 2010, 345, 2390-2400.
(7) Zhang, Z., Anal. Chem. 2004, 76, 3908-3922.
(8) Domon, B.; Costello, C. E., Glycoconjugate J. 1988, 5, 397-409.
(9) Dallinga, J. W.; Heerma, W., Biol. Mass Spectrom. 1991, 20, 215-231.
(10) Zaia, J., Mass Spectrom. Rev. 2004, 23, 161-227.
(11) Thomson, B. A. Apparatus and method for MSnth in a tandem mass spectrometer system. U.S. Patent 7,145,133 B2, December 5, 2006.
(12) Thomson, B. A.; Jolliffe, C. L. Spectrometer with axial field. U.S. Patent 5,847,386, December 8, 1998.
(13) Loboda, A.; Krutchinsky, A.; Loboda, O.; McNabb, J.; Spicer, V.; Ens, W.; Standing, K., Eur. J. Mass Spectrom. 2000, 6, 531-536.
(14) Collings, B. A., J. Am. Soc. Mass Spectrom. 2007, 18, 1459-1466.
(15) Morris, M.; Pierre, T.; B., R. K., J. Am. Soc. Mass Spectrom. 1994, 5, 1042-1063.
(16) Carroll, J. A.; Willard, D.; Lebrilla, C. B., Anal. Chim. Acta 1995, 307, 431-447.
75
CHAPTER 4 Z1 IONS AS DIAGNOSTIC IONS FOR LINKAGE DETERMINATION
4.1 Introduction
In terms of linkage determination, disaccharides have been extensively studied as
model systems in the positive ion mode as metal ion adducts1-6 and in the negative ion
mode as deprotonated molecular ions7-11 or anion adducts of the molecules.12 Based on
the patterns of A-type product ions, such as m/z 221, 251, 263, and 281, formed by cross-
ring cleavages within the reducing sugar via various dissociation methods, the linkage
positions can be determined.1-15 Furthermore, the relative intensities of A ions as well as
the cleavage products from either side of the glycosidic oxygen provided additional
evidence for linkage assignment in the negative ion mode.11 Linkage determination for
oligosaccharides takes the same approach by detecting a certain combination of A ions.
Data analysis, however, can be much more challenging for oligosaccharides due to the
co-existence of isomeric and isobaric peaks from MS/MS, potentially derived from either
end of the molecule. Permethylation16 or reducing-end modification17 have therefore been
utilized to simplify the situation by providing mass discrimination of the product ions.
Currently, MS2 employing collision-induced dissociation (CID) is still the most widely
used strategy for linkage determination. However, depending on the nature of analytes
(trisaccharides or larger) and CID conditions, key diagnostic ions (i.e. A type ions) may
be missing, leading to either miss-assigned or un-assigned linkage positions.5,8,9 In order
76
to maximize the structural information inherent in fragmentation patterns using MS2 for
oligosaccharide analysis, different activation/dissociation methods have been
investigated, including infrared multiphoton dissociation (IRMPD),18 electron capture
dissociation (ECD),19,20 electron-induced dissociation (EID),21 electron detachment
dissociation (EDD),22 and electron excited dissociation (EED).23 The application of the
above methods has been limited to research groups owing these instrumental capabilities.
Overall, MS2, the simplest MS/MS, has advantages such as good sensitivity, high
throughput, and simplicity in performance and instrumentation. However, structural
information to firmly establish anomeric configurations and stereochemistries of
individual monosaccharides and some of their linkages within oligosaccharides often
cannot be obtained using MS2 alone.
In this chapter, we describe an MSn approach to extract linkage information from
linear oligosaccharides together with their locations within the molecules with greater
confidence than is currently possible. We discovered that CID of Z1 ions derived from the
reducing sugar of deprotonated disaccharides showed distinct fragmentation patterns
virtually solely based on their linkage types and not significantly affected by the sugar
unit identities or their anomeric configurations. An MSn CID (n = 3-5) strategy was
developed that permitted linkages to be determined more confidently within a linear
oligosaccharide by decomposing the oligomer in the gas-phase into a set of overlapping
disaccharide structures with known origins within the oligosaccharide. Z1 ions were then
optimized for each disaccharide subunit, and repeatable CID spectra were obtained for Z1
ions derived from independent disaccharides. Spectral similarity scores were employed
for linkage determination via spectral matching of the thus obtained Z1 CID data to the
77
standard disaccharide database. At a stage of proof-of-principle, this approach was
successfully demonstrated with two trisaccharides and a pentasaccharide.
4.2 Experimental
4.2.1 Materials
A list of 17 disaccharides and 3 oligosaccharides is shown in Table 4.1. All
samples were purchased from commercial sources (indicated by the superscripts in Table
4.1) and used without further purification. 4-Aminobenzoic acid (ABA), ethyl 4-
aminobenzoate (ABEE), H218O (97 atom %), chloroform, sodium cyanoborohydride,
acetic acid, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, Inc.
(St. Louis, MO, USA). Detailed procedures of 18O-labeling24 and reductive amination25,26
of reducing saccharides with ABA and ABEE were described in the previous chapters
(18O-labeling in chapter 2 and reductive amination in chapter 3). α1-2,3 Mannosidase
with G6 reaction buffer and BSA was purcharsed from New England Biolabs, Inc.
(Ipswich, MA, USA). Digestion condition was followed the procedure from
manufacture.
78
Table 4.1 List of disaccharides and oligosaccharides being studied.
Analytes were purchased from: a Carbosynth, Ltd. (Berkshire, UK) and b Sigma-Aldrich, Inc. (St. Louis, MO, USA).
4.2.2 Reducing End Modification (Amination without Reduction)
Similar to the reductive amination except for using sodium cyanoborohydride was
used. Briefly, disaccharides or oligosaccharides (0.5 mg) were dissolved in 10 μL of a
solution containing 0.15 M ABA, ABEE, or APA in acetic acid: DMSO (3:7, v/v), and
the solution was incubated at 60 °C for 10-12 h. The solvent was removed under vacuum
(Speed-Vac, LABCONCO, Kansas City, MO) and the residue was dissolved in 300 μL
water followed by an addition of 300 μL chloroform. The aqueous phase containing the
derivatized sugar was collected after vortex mixing. This step was repeated with another
300 μL of water for extraction and the aqueous phases were combined and vacuum dried.
79
The dried sample was dissolved in 100 μL DMSO and 900 μL methanol. This solution
was further diluted 100-fold in methanol prior to MS analysis.
4.2.3 Mass Spectrometry
All samples were analyzed in the negative ion mode on a hybrid triple
quadrupole/linear ion trap mass spectrometer (QTRAP4000 Applied Biosystems,
Toronto, Canada) equipped with a home-built nanoelectrospray ionization (nanoESI)
source. A schematic diagram of the instrument is shown in Scheme 2.2. Two types of low
energy collisional activation were accessible on this instrument, i.e. beam-type CID and
ion trap CID (via on-resonance single frequency excitation). In beam-type CID, the
precursor ions were isolated in Q1, accelerated to the Q2 collision cell for collisional
activation, and all products were analyzed in the Q3 linear ion trap. The collision energy
(CE, in the lab frame) was defined by the difference of the dc rod offsets between Q0 and
Q2. For ion trap CID, the precursor ions were isolated in the Q3 linear ion trap via
RF/DC mode and a dipolar excitation was applied at a Mathieu q value of 0.23. MS2 CID
experiments were carried out by either beam-type or ion trap CID. Higher stages of CID,
i.e., MS3 to MS5, were carried out by ion trap CID in Q3. MSn (n≥3) experiments were
performed through access to the original method table. Analyst 1.5 software was used for
instrument control, data acquisition, and processing. The typical parameters of the mass
spectrometer used in this study are listed in Supporting Information. The standard
spectra of Z1 ions (either m/z 163 from 18O-labeled glucose homodimers or m/z 161 from
intact glucose homodimers) were generated based on the average of 6 spectra over a 6-
month period. Standard deviations of peak heights were calculated for major 14
80
fragments including m/z 73, 83, 89, 97, 101, 103, 113, 115, 131, 133, 135, 143, and 145
for CID spectra of m/z 163 and m/z 73, 83, 87, 89, 97, 101, 103, 113, 115, 125, 131, 133,
143, and 161 for CID spectra of m/z 161. These peaks were also used to calculate spectral
similarity scores.
4.2.4 HPLC
ABEE modified branched mannose 5 and exoglycosidase digested mannose 3
were separated using Agilent 1200 series HPLC system (Agilent Technologies, Santa
Clara, CA, USA). Separation was carried out on a HILIC (PolyLC, Inc., Columbia, MD,
USA) at a flow rate of 1 mL/min with a linear gradient of 20-35% solvent A in 30 min.
Solvent A was a mixture of 5mM ammonium acetate in water and solvent B contained 5
mM ammonium acetate in 100% acetonitrile. The eluent was detected at a wavelength of
305 nm.
4.3 Results and Discussion
4.3.1 Disaccharide Linkage Analysis Based on MS3 CID of Z1 Ions
Disaccharides, the smallest substructures of oligosaccharides containing linkage
information between two complete sugar molecules were used as model systems for
evaluating mass spectra of product ions that may yield detailed information about the
linkage positions. Initially, we used 18O-labeling at the carbonyl oxygen of 15
disaccharides to introduce a mass discrimination between X, Y, and Z vs. A, B and C
81
product ions,27 recognizing that some of the ions might be isomeric and need to be
resolved as isotopomers. MS2 beam-type CID spectra of m/z 343 precursor ions of
glucose homodimers ( -anomeric configurations) with four different linkage positions
are shown in Figure 4.1. In these spectra, m/z 223, 253, 265, and 283 ions have
incorporated 18O (compared to the non-labeled data), suggesting that these fragments
contained a labeled component derived from the reducing sugar unit. Depending on the
disaccharide, some of the other product ions were observed as pairs varying in mass by 2
m/z units, the higher m/z isomer not having been observed in unlabeled disaccharides. Of
pertinence to this paper, peaks at m/z 161 and 163 were observed, indicative of two
isomers which would have been indistinguishable without 18O-labelling. A zoomed-in
region showing m/z 161 and 163 product ions is presented in insets for each linkage
position. Ions at m/z 163 should be Z1 ions, formed by cleavage at the reducing side of
the glycosidic oxygen. This ion species is unlikely to be formed as a sequential water loss
from Y1 ions (m/z 181), which we have not observed for MS2 CID of 18O-labeled
disaccharides. The assignment is also supported by the fact that MS3 CID of m/z 163
product ions showed totally different fragmentation patterns as compared to those
generated from H2O loss of 18O-labeled monosaccharides (data not shown). Furthermore,
MS3 CID of m/z 161 product ions (Figure 4.2) from the 18O-labeled disaccharides yielded
mass spectra that were very different from those derived from the m/z 163 ions,
suggesting that they have different origins and structures. These above results indicated
that from disaccharides, m/z 163 product ions were generated by dissociation pathways
unique to the substitution position of the reducing sugar.
82
Figure 4.1 MS2 beam-type CID of 18O-labeled α-D-glucose homodimers, m/z 343 ([M-H]-), with different linkage positions: (a) 1-2, CE: 8 V; (b)1-3, CE: 12 V; (c) 1-4, CE: 10 V; and (d) 1-6, CE: 10 V. Insets in each of the spectra show the expanded region covering m/z 161 and 163.
161 163
265
179 343325223
161143119
101
161 163
179
343
221
323143101
161 325
281
113
251
113
163119
0
50
100
Rel
. Int
. (%
)
60 100 140 180 220 260 300 340m/z
(d) α-D-Glcp-(1-6)-D-Glc-18O
(a) α-D-Glcp-(1-2)-D-Glc-18O
(c) α-D-Glcp-(1-4)-D-Glc-18O
0
50
100
Rel
. Int
. (%
)
60 100 140 180 220 260 300 340m/z
0
50
100
Rel
. Int
. (%
)
60 100 140 180 220 260 300 340m/z
343
115
163343
179 283145113
22714389
161
253281
325
161 163
(b) α-D-Glcp-(1-3)-D-Glc-18O
0
50
100R
el. I
nt. (
%)
60 100 140 180 220 260 300 340m/z
343
343
343
163
179263 283103
161 161 163
281145143
343
83
Figure 4.2 MS3 ion trap CID of m/z 161 product ions derived from 18O-labeled disaccharides ( -anomeric configuration) with different sugar identities and linkage positions: (a) α-D-Glcp-(1-2)-D-Glc, (b) α-D-Galp-(1-3)-D-Gal, (c) α-D-Glcp-(1-3)-D-Glc, (d) α-D-Manp-(1-4)-D-Man, (e) α-D-Glcp-(1-4)-D-Glc, (f) β-D-Galp-(1-4)-D-Man, (g) α-D-Glcp-(1-6)-D-Glc, and (h) α-D-Galp-(1-6)-D-Glc.
113
143131
161125101
(a) α-D-Glcp-(1-2)-D-Glc-18O
60 80 100 120 140 160m/z0
100R
el. I
nt. (
%)
50
343
161
(e) α-D-Glcp-(1-4)-D-Glc-18O113
131143
161125101838760 80 100 120 140 160m/z
0
100
Rel
. Int
. (%
)
50
343
161
113
131143
10183 16112587
(c) α-D-Glcp-(1-3)-D-Glc-18O
60 80 100 120 140 160m/z0
100
Rel
. Int
. (%
)
50
343
161
113
143131
16187 12510183
(g) α-D-Glcp-(1-6)-D-Glc-18O
60 80 100 120 140 160m/z0
100
Rel
. Int
. (%
)
50
343
161
60 80 100 120 140 160m/z0
100
Rel
. Int
. (%
)
50
113
143161131
60 80 100 120 140 160m/z0
100
Rel
. Int
. (%
)
50
113
131143
101 161
(b) α-D-Galp-(1-3)-D-Gal-18O343
161
(d) α-D-Manp-(1-4)-D-Man-18O343
161
73 8387
83 87
(f) β-D-Galp-(1-4)-D-Man-18O
(h) α-D-Galp-(1-6)-D-Glc-18O 60 80 100 120 140 160m/z
0
100R
el. I
nt. (
%)
50
113
131
14316110183
60 80 100 120 140 160m/z0
100
Rel
. Int
. (%
)
50
113
143131
161101
343
161
343
161
84
MS3 ion trap CID spectra of m/z 163 ions (Z1 ions) were collected for 15 18O-
labele disaccharides. Figure 4.3 shows the data sets obtained from glucose homodimers
with 4 different linkage positions. Note that each panel is an average of 6 spectra (3
spectra each from α- and β-anomeric configurations) collected over 6 months. The error
bars on the major peaks represent the standard deviation of the six spectra. In the standard
spectra, CE was controlled to reduce the parent ion intensity to 18% of the base peak,
which was found to provide good reproducibility of the fragmentation patterns. Clearly,
CID of m/z 163 produces distinct fragmentation patterns characteristic of the linkage
positions. There are 13 common peaks (>3% relative intensity) observed from these
spectra, however with quite different relative intensities for different linkage positions.
These include ions at m/z 73, 83, 89, 97, 101, 103, 113, 115, 131, 133, 135, 143, and 145.
For the 1-2 linkage (Figure 4.3a), the base fragment peak is at m/z 103 and other
signature peaks include m/z 101, 115, 133, 135, and 145. We did notice that the α- and β-
anomers showed a small difference in the CID fingerprints, which was reflected by the
larger standard deviations of peak intensities at m/z 115 and 145 (22% and 9%),
respectively. These two peaks are of higher relative intensities in the β-anomer as
compared to the α-anomer. The comparisons of MS3 CID of m/z 163 from the α- and β-
anomers can be found in Figure 4.4. For the 1-3 linkage (Figure 4.3b), the base peak
shifts to m/z 115 and peaks at m/z 115 and 145 are relatively abundant. All other fragment
ions are present with relatively low intensities (<5%). The signatures of the 1-4 linkage
(Figure 4.3c) include dominant fragments at m/z 83 and 103 (base peak), while other
major fragments (m/z 73, 89, 97, 115, 133, and 145) have similar intensities around 20-
30%. The 1-6 linkage (Figure 4.3d) produces a relatively simple fragmentation spectrum
85
with m/z 101 as the base peak, while other fragments (m/z 103, 131, and 135) show
relative intensities lower than 10%. Note that these fragmentation patterns are very
distinctive according to the linkage positions and repeatable as reflected by the relatively
small standard deviations of all major peaks. These characteristics of Z1 fragmentation
make it suitable as a diagnostic ion for linkage determination. With 18O-labeling on the
reducing sugar carbonyl group, dissociation profiles of the m/z 163 ion enable the linkage
position to be determined with high confidence.
Another interesting aspect that we discovered from CID of Z1 ions from 15
disaccharides was that neither the stereochemistry (identity) of the sugar units nor the
anomeric configuration (except for the 1-2 linkage discussed above) had any noticeable
effects on the fragmentation patterns. CID of Z1 ions from 7 disaccharides other than
glucose homodimers were compiled and shown in Figure 4.5. For example, the five
different 1-4 linked disaccharides (Figure 4.5a to e) showed almost identical
fragmentation patterns to that of the 1-4 linked glucose homodimer (Figure 4.3c). This
was consistently observed for other disaccharides containing 1-3 and 1-6 linkages (for
instance, compare Figure 4.5f to Figure 4.3b and Figure 4.5g to Figure 4.3d). Although
this phenomenon precludes obtaining stereo-structural information from the m/z 163 (Z1)
product ion (i.e., identity and anomeric configuration) for the reducing sugar unit, it is
advantageous for linkage determination due to the simplification of producing data from
standards and in making comparisons to unknowns for spectral analysis.
86
Figure 4.3 Averaged MS3 ion trap CID data of Z1 ions (m/z 163) derived from 18O-labeled glucose homodimers with different linkage positions: (a) 1-2, (b) 1-3, (c) 1-4, and (d) 1-6. The error bars represent standard deviations of peak intensities based on 6 averaged spectra (3 spectra each from - and -anomers) obtained over a 6-month period.
(a) 1-2 linkage343
163
60 80 100 120 140 160 1800
100
Rel
. Int
., %
m/z
103
133101
163135
115
145
(b) 1-3 linkage115
145
163
343
163
60 80 100 120 140 160 1800
100
Rel
. Int
., %
m/z(c) 1-4 linkage
343
163
60 80 100 120 140 160 1800
100
Rel
. Int
., %
m/z
10383
73 133 16397 11589 145
(d) 1-6 linkage101
163131103 135
343
163
60 80 100 120 140 160 1800
100
Rel
. Int
., %
m/z
87
Figure 4.4 MS3 CID spectra of m/z 163 product ions from (a) α-D-Glcp-(1-2)-D-Glc-18O and (b) β-D-Glcp-(1-2)-D-Glc-18O. Additional peaks (m/z 115 and 145) were found in the β configuration.
-H -β-D-Glcp-(1-2)-D-Glc-18O
103
133115
101 135145 163113 131 14360 80 100 120 140 160
m/z0
100
Rel
. Int
. (%
)
50
-H -α-D-Glcp-(1-2)-D-Glc-18O
(a) 343
163
60 80 100 120 140 160m/z
0
100
Rel
. Int
. (%
)
50
103
133101
163135
(b)
113 143
343
163
88
Figure 4.5 MS3 ion trap CID of m/z 163 product ions from (a) α-D-Manp-(1-4)-D-Man-18O, (b) β-D-Galp-(1-4)-D-Glc-18O, (c) α-D-Galp-(1-4)-D-Gal-18O, (d) β-D-Galp-(1-4)-D-Man-18O, (e) α-D-Manp-(1-4)-D-Glc-18O, (f) α-D-Galp-(1-3)-D-Gal-18O, and (g) α-D-Galp-(1-6)-D-Glc-18O.
60 80 100 120 140 160m/z
0
100
Rel
. Int
. (%
)
50
60 80 100 120 140 160m/z
0
100
Rel
. Int
. (%
)
50
60 80 100 120 140 160m/z
0
100
Rel
. Int
. (%
)
50
60 80 100 120 140 160m/z
0
100
Rel
. Int
. (%
)
50
60 80 100 120 140 160m/z
0
100
Rel
. Int
. (%
)
50
115
14510383 163113
101
163131103 135
(f) α-D-Galp-(1-3)-D-Gal-18O
(a) α-D-Manp-(1-4)-D-Man-18O103
83
73 163115 13397 14589
(b) β-D-Galp-(1-4)-D-Glc-18O10383
73133 145 1631159789
(e) α-D-Manp-(1-4)-D-Glc-18O10383
73115 133 145 1639789
60 80 100 120 140 160m/z
0
100
Rel
. Int
. (%
)
50
10383
73133 145 1631159789
343
163
(c) α-D-Galp-(1-4)-D-Gal-18O
60 80 100 120 140 160m/z
0
100
Rel
. Int
. (%
)
50
10383
73133 1631451159789
343
163
(d) β-D-Galp-(1-4)-D-Man-18O
(g) α-D-Galp-(1-6)-D-Glc-18O
343
163
343
163
343
163
343
163
343
163
89
4.3.2 The Effect of CID Conditions on the Formation of Z1 Ions
The data in Figure 4.3 demonstrate that CID of Z1 ions (m/z 163) can be used for
linkage determination. Note that the formation of Z1 ions was typically accompanied by
its structural isomer, ions at m/z 161 (insets of Figure 4.1). Collisional activation of the
m/z 161 ions from 18O-labeled disaccharides did not provide any distinguishable
fragmentation patterns according to their sugar unit identities, anomeric configurations,
or linkage types within the disaccharides. Although reducing-end labeling with 18O can
be used to clearly distinguish Z1 ions vs. m/z 161 ions, it was also highly desirable to
optimize the formation of Z1 ions while minimizing the abundance of their structural
isomer(s) (m/z 161) when studying native disaccharides or disaccharide units formed by
gas-phase dissociation of oligosaccharides. We investigated the effect of CID (beam-type
and ion trap) conditions on the formation of Z1 ions vs. their structural isomers using 18O-
labeled glucose homodimers (1-2, 1-3, 1-4, and 1-6 linkages and both α and β anomers).
The results are summarized in Figure 4.6 showing one anomer type as an example for
each linkage because the same trend of fragmentation behavior was observed for both
anomeric configurations. These spectra were collected using a wide isolation window
(around 5 Da) to observe both m/z 161 and 163 ions. Data from three different CEs (low,
medium, and high) under beam-type CID (left column) and ion trap CID (right column)
are compared side-by-side.
The best condition for forming Z1 ions from 1-2 linked disaccharides was the
lower energy ion trap CID (Figure 4.6a, 5 mVpp), from which m/z 163 accounted for 80%
of the summed intensities of m/z 161 and 163. Higher CE ion trap CID (7.5 and 10 mVpp)
significantly increased the relative intensity of m/z 161. Note that when beam-type CID
90
was employed, m/z 161 was the dominant peak, while Z1 ions (m/z 163) were not
detected above noise level. On the other hand, 1-3 and 1-4 linked disaccharides showed
dominant formation of Z1 ions relative to m/z 161 under both beam-type and ion trap CID
conditions (Figure 4.6b and c, respectively). For 1-6 linked disaccharides, the best
conditions for the formation of Z1 ions were found using lower CEs for both beam-type
and ion trap CID as shown in Figure 4.6d. The data in Figure 4.6 clearly demonstrate that
the formation of m/z 161 and 163 is strongly affected by collisional activation conditions
and the best common condition to obtain relatively pure Z1 ion for all the linkage types
was to use ion trap CID with relatively low energy (around 5 mVpp). It should be noted
that even using these optimized conditions, the purity of Z1 ions from 1-2 linked
disaccharides was about 80%. After determining the CID conditions for preferential
formation of Z1 ions, the spectra in Figure 4.3 were re-collected for native unlabeled
glucose homodimers with relatively low energy ion trap CID. The data are shown in
Figure 4.7. Characteristic and highly reproducible fragmentation patterns were obtained,
which were almost identical to the CID of Z1 ions derived from 18O-labeled samples. The
data indicated that under the optimized CID conditions, the existence of small impurities
within the Z1 ions did not significantly affect their distinct fragmentation patterns. These
m/z 161 CID spectra were later used as standard spectra for Z1 ion fragmentation derived
from native disaccharides or disaccharide substructures of oligosaccharides.
We also performed MS3 CID of Z1 ions from two fructose containing
disaccharides (α-D-Glcp-(1-3)-D-Fru and β-D-Galp-(1-4)-D-Fru) with and without 18O-
labeling. Fructose has a ketone group at C2 position and 18O is incorporated in hydroxyl
group at C2 position as compared to the C1 position for aldohexoses. Collisional
91
activation of Z1 ions from the two 18O-labeled fructose containing disaccharides showed
characteristic fragmentation patterns of 1-3 and 1-4 linkages (Figure 4.8a and c),
respectively. The fragment fingerprint patterns are identical to the data obtained from the
1-3 and 1-4 linked glucose homodimers (Figure 4.3b and c). The 1-4 linked fructose
containing disaccharide data (Figure 4.8c), however, showed 2 m/z shifts for fragments
such as m/z 75, 87, 99, 117, and 135, as compared to the aldohexose containing
disaccharide data (Figure 4.3c). The mass shifts are likely contributed by the difference
of 18O-labeling position between an aldohexose and a ketohexose. Using the optimized
conditions for Z1 ion formation (ion trap CID, 5 mVpp), MS3 CID of Z1 ions of the two
unlabeled fructose containing disaccharides also showed characteristic fragmentation
patterns of 1-3 and 1-4 linkages (Figure 4.8b and d). These results confirmed that MS3
CID of Z1 ions can be applied to disaccharide units containing fructose for determination
of linkage position.
92
Figure 4.6 The effect of CID conditions on the formation of Z1 (m/z 163) vs. its structural isomers (m/z 161). Data were obtained from 18O-labeled glucose homodimers: (a) α-D-Glcp-(1-2)-D-Glc, (b) β-D-Glcp-(1-3)-D-Glc, (c) β-D-Glcp-(1-4)-D-Glc, and (d) α-D-Glcp-(1-6)-D-Glc.
161 163 161 163
510
15
5
2030
m/z m/z
(d) 1-6 linkage
Beam-type CID Ion trap CID
(c) 1-4 linkage
161 163 161 163
525
35
5
12.520
m/z m/z
161 163 161 163
57.5
10
m/z m/z
161 163 161 163
5
1015
5
7.5
10
m/z m/z
(b) 1-3 linkage
(a) 1-2 linkage
5
1015
93
Figure 4.7 Averaged MS3 ion trap CID data of Z1 product ions (m/z 161) derived from native unlabeled glucose homodimers with different linkage positions: (a) 1-2, (b) 1-3, (c) 1-4, and (d) 1-6. The error bars show standard deviation of peak intensities based on 6 averaged spectra (3 spectra each from - and -anomers) obtained over a 6-month period. Ion trap CID with 5 mVpp was used for MS2 CID of m/z 341 step for (a) through (d).
(a) 1-2 linkage341
161
60 80 100 120 140 160 1800
100
Rel
. Int
., %
m/z(b) 1-3 linkage
341
161
60 80 100 120 140 160 1800
100
Rel
. Int
., %
m/z(c) 1-4 linkage
341
161
60 80 100 120 140 160 1800
100
Rel
. Int
., %
m/z
10183
73 133 16197 11587 143
(d) 1-6 linkage101
161
341
161
60 80 100 120 140 160 1800
100
Rel
. Int
., %
m/z
101
131161133
113
143
143161
113
131
94
Figure 4.8 MS3 ion trap CID of the Z1 ion from 18O-labeled fructose containing disaccharides and MS3 ion trap CID of the Z1 ion generated by ion trap CID with low energy from native unlabeled fructose containing disaccharides, (a) α-D-Glcp-(1-3)-D-Fru-18O, (b) α-D-Glcp-(1-3)-D-Fru, (c) β-D-Galp-(1-4)-D-Fru-18O, and (d) β-D-Galp-(1-4)-D-Fru.
4.3.3 Linkage Determination for Oligosaccharides via MSn CID of Z1 Ions
Given that linkage positions can be determined from MS3 CID of Z1 ions from
deprotonated disaccharides, we further extended this method to linear oligosaccharides.
The approach involved optimizing glycosidic bond cleavages during early stages of MSn
so that an ordered set of overlapping disaccharide substructures could be formed,
generating Z1 ions from each disaccharide unit, and finally obtaining the fragmentation
fingerprints of Z1 ions for linkage determination. The concept is illustrated with a
pentasaccharide in Scheme 4.1, with the numbering of the sugar units starting from the
(b) α-D-Glcp-(1-3)-D-Fru
60 80 100 120 140 160m/z
0
100
Rel
. Int
. (%
)
50
113
143
161
341
161
(d) β-D-Galp-(1-4)-D-Fru
60 80 100 120 140 160m/z
0
100
Rel
. Int
. (%
)
50
10183
73133 161115 14397
341
161
87
(a) α-D-Glcp-(1-3)-D-Fru-18O
60 80 100 120 140 160m/z
0
100
Rel
. Int
. (%
)
50
115
145
163
(c) β-D-Galp-(1-4)-D-Fru-18O
60 80 100 120 140 160m/z
0
100
Rel
. Int
. (%
)
50
10383
75163135117 14587 99
343
163
343
163
95
reducing-end (sugar 1). The key steps include: 1) dissociation of a precursor derivatized
at the reducing-end (M, Scheme 1) to a ladder of smaller oligosaccharides, each
successively one sugar unit shorter, containing the reducing-end tag (Ym ions); 2)
generation of disaccharide fragments (C2 ions) from each of the Ym ions; 3) dissociation
of the disaccharide units to form Z1 ions; 4) CID of Z1 ions and 5) spectral matching of
the Z1 CID data to the standard database (derived from disaccharides, Figure 4.3 or
Figure 4.7) for linkage determination. Linkage positions between the first and the second
sugar units could be directly obtained from 18O-labled oligosaccharides via MS3 CID of
m/z 163. The above approach has the advantage of knowing the exact origin of each
fragment with respect to its initial position within the oligomer. The reducing-end
modification M introduces a mass distinction between Y and C ions. In addition, the
group used to derivatize the reducing- end bears a negative charge and enabled the
selection of the ladder of charged Ym ions after MS2. Yet, through charge-transfer, it
permitted disaccharide (C2) ions to be formed and isolated. For example, to obtain
linkage information between sugar units 3 and 4, the following MS5 is needed: [[M-H]-
Y4 C2 Z1 fragments] (shown in Scheme 4.1).
96
Scheme 4.1 The MSn approach for linkage determination of an oligosaccharide. “M” stands for reducing-end modification.
Two trisaccharides and one pentasaccharide were used to test the MSn approach
for extracting linkage information using this approach. The data in Figure 4.9 show a
series of experiments (MS2 to MS4) using 18O-labeled α-D-Glcp-(1-6)-α-D-Glcp-(1-4)-
D-Glc (Mw: 506 Da). In order to determine the linkage positions of linkages 1 and 2
(counting from the reducing- end), the following experiments were needed: MS3 CID
[505 163 fragments] and MS4 CID [505 341 161 fragments]. MS2 CID (CE =
15 V, beam-type) of the deprotonated molecular ions ([M-H]-) is shown in Figure 4.9a, in
which the formation of Z1 ions (m/z 163) and C2 ions (m/z 341) can be clearly seen. MS3
CID of Z1 ions (ion trap CID, 27 mVpp, Figure 4.9c) showed an almost identical spectrum
to the 1-4 linkage standard spectrum (Figure 4.3c). Therefore, linkage 1 can be
confidently identified as a 1-4 linkage. Note that the linkage could be assigned directly
from the m/z 163 ion derived from the labeled trisaccharide without requiring prior
2 1O M3O4 OO5
2 1O M3O4 OHO
3O4 OHHO
3 OH MS5 CID
Unique Dissociation
Pattern
DisaccharideStandardDatabaseSpectral Matching
LinkagePosition
Gas
-Pha
seD
isso
ciat
ion
OO 4OO 4Y4
OO3C2
3OO
Z1
97
dissociation and isolation of the m/z 343 reducing-end disaccharide. This is particularly
worth emphasizing as the m/z 505 precursor ion gave rise to negligible quantities of the
m/z 343 product ion (Figure 4.9a), thus information could not be obtained for this linkage
either from the direct dissociation pattern of its reducing disaccharide (m/z 343) or from
the 163 product ion that may have been derived from it. Another important point is that
since m/z 161 ions are also formed in MS2 CID (due to sequential fragmentation of C1
ions), it is necessary to use m/z 163 ions to achieve correct linkage information for the
reducing end. Low energy MS3 CID (ion trap, 5 mVpp) of C2 ions (m/z 341, Figure 4.9a)
produced a reasonable intensity of m/z 161 ions (Figure 4.9b). Note that the best CID
conditions characterized from disaccharide studies (ion trap CID, 5 mVpp, 200 ms) were
used to favor the formation of Z1 ions relative to their structural isomers. Indeed, MS4
CID (ion trap, 35 mVpp) of m/z 161 showed a fragmentation pattern characteristic of the
1-6 linkage position (compare Figure 4.9d to the standard spectrum in Figure 4.7d). For
the other trisaccharide sample, 18O-labeled α-D-Galp-(1-3)-β-D-Galp-(1-4)-D-Gal, the
same set of MS3 and MS4 experiments were required to obtain information for linkages 1
and 2 : ([505 163 fragments] and [505 341 161 fragments]). Abundant Z1 and
C2 ions were formed under MS2 beam-type CID (CE = 15 V) as shown in Figure 4.10a.
MS3 CID (ion trap, 27 mVpp) of the Z1 ion clearly showed the distinct fragmentation
pattern of a 1-4 linkage position (Figure 4.9e). MS3 CID (ion trap, 5 mVpp) of C2 ions
(m/z 341) (Figure 4.10b) produced abundant Z1 ions (m/z 161). MS4 CID (ion trap, 35
mVpp) of the Z1 ions (m/z 161) optimized in abundance is shown in Figure 4.9f. The
fragmentation pattern matched well to the 1-3 linkage standard spectrum (Figure 4.7b)
and allowed confident assignment of this linkage position.
98
Figure 4.9 MSn CID spectra for the determination of linkage types within trisaccharides. α-D-Glcp-(1-6)-α-D-Glcp-(1-4)-D-Glc-18O: (a) MS2 beam-type CID: [505 fragments], (b) MS3 ion trap CID: [505 341 fragments], (c) MS3 ion trap CID: [505 163 fragments], (d) MS4 ion trap CID: [505 341 161 fragments]. α-D-Galp-(1-3)-β-D-Galp-(1-4)-D-Gal-18O: (e) MS3 ion trap CID: [505 163 fragments] and (f) MS4 ion trap CID: [505 341 161 fragments].
(b)
60 140 220 300
179
323281
161221
341143311
m/z
2510
50
100
0
50
100
Rel
. Int
. (%
)
(a)
100 200 300 400 500
341 505
383179
323221143
161113
101 281 425
m/z
161 163
505
(c) 505
163
505
341
505
341
161
(d)
60 80 100 120 140 160m/z
101
161131
C2
C1Z1
α-D-Glcp-(1-6)-α-D-Glcp-(1-4)-D-Glc-18O, m/z 505
The parent ion The product ion for CID in the next stage
103
83
145133115 163738997
60 80 100 120 140 160m/z
0
50
100
Rel
. Int
. (%
)R
el. I
nt. (
%)
(e)
60 80 100 120 140 160m/z
0
50
100
Rel
. Int
. (%
)
83103
73 115 133145 1638997
505
163(f)
60 80 100 120 140 160m/z
113
143 161
505
341
161
α-D-Galp-(1-3)-β-D-Galp-(1-4)-D-Gal-18O, m/z 505
99
Figure 4.10 MS2 and MS3 CID spectra from α-D-Galp-(1-3)-β-D-Galp-(1-4)-D-Gal-18O.(a) MS2 beam-type CID: [505 fragments], and (b) MS3 ion trap CID: [829 341 fragments].
In order to determine the individual linkages within the glucose homopentamer,
[β-D-Glcp-(1-4)]4-D-Glc using the MSn approach, formation of the following ions at the
MS2 stage was a prerequisite from the 18O-labeled sample: m/z 163 (Z1), 341 (C2), 505
(Y3), and 667 (Y4). MS2 CID of the deprotonated molecular ion (m/z 829, Figure 4.11a)
produced major peaks at m/z 341 (C2), 503 (C3), 665 (C4), and also a small amount of Z1
ions (m/z 163). However, no Y ions were detected above the noise level, which made it
impossible to conduct MSn to determine linkages 2 and 3. Reducing-end derivatization
with different types of functional groups has been shown to alter the fragmentation
patterns of oligosaccharides and generates X, Y, and Z product ions of
oligosaccharides.17 We tried reductive amination with ABA and ABEE at the reducing
α-D-Galp-(1-3)-β-D-Galp-(1-4)-D-Gal-18O, m/z 505
0
50
100
Rel
. Int
. (%
)
(a)
100 200
505161505
341
113 179
443143
323 425
163 C2Z1
C1
300 400 500
(b)
60 140 220 300m/z
0
50
100 505
341
161 341
179
113143 323281
Rel
. Int
. (%
)342
The parent ion The product ion for CID in the next stage
100
sugar to form the reduced form of the corresponding Schiff base. Only data from ABEE
are discussed here since higher intensity of the desired ions were observed. The full series
of MS2 to MS5 spectra of ABEE modified [β-D-Glcp-(1-4)]4-D-Glc is shown in Figure
4.12. MS2 beam-type CID of the doubly deprotonated molecular ion, m/z 487 (Figure
4.12a), produced a variety of C and Y ions: m/z 341 (C2), 406 (Y42-), 503 (C3), 652 (Y3),
and 814 (Y4). The formation of reasonable intensities of Y3 and Y4 allowed further stages
of MS/MS. Finally, the following MSn CID sequences were employed for the
determination of linkages 1-4 individually: linkage 1, MS3 CID [829 163 fragments]
from the 18O-labeled sample; linkage 2, MS5 CID [487(2-
) 652 341 161 fragments] from the ABEE labeled sample; linkage 3, MS5 CID
[487(2-) 406(2-) 341 161 fragments] from the ABEE labeled sample; and linkage
4, MS4 CID: [829 341 161 fragments] from 18O-labeled sample. These four spectra
all showed characteristic fragmentation patterns of the 1-4 linkage position, allowing the
linkage position to be confidently assigned as 1-4. The data for individual stages of
MS/MS within each sequence of MSn CID can be found in Figure 4.11 (for the 18O-
labeled sample), and 4.12 (for the ABEE labeled sample).
101
Figure 4.11 MS2 and MS4 CID spectra from [β-D-Glcp-(1-4)]4-D-Glc-18O. Experimental step for linkage 1 analysis is [829 163 fragments], (a) MS2 beam-type CID: [829 fragments], (b) MS3 ion trap CID: [829 163 fragments]. Experimental step for linkage 2 analysis is [829 341 161 fragments], (c) MS3 ion trap CID: [829 341 fragments], and (d) MS4 ion trap CID: [829 341 161 fragments].
--HZ1, m/z 163C2, m/z 341
60 140 220 300 380m/z
161
341
179281
(c)
0
50
100 83101
73 16113311597 14387 125
(d)
60 80 100 120 140 160m/z
0
50
100829
341(b)83 103
11589 1639775 13573 145
117
60 80 100 120 140 160m/z
0
50
100
Rel
. Int
. (%
)
829
163
829
341
161
(a)829
0
50
100
Rel
. Int
. (%
)
200 600 800m/z400
665503
341
587 829749425485
281323
161
161 163
C2
C3C4
Z1
[β-D-Glcp-(1-4)]4-D-Glc-18O, m/z 829
The parent ion The product ion for CID in the next stage
102
Figure 4.12 MS2 to MS5 CID spectra from ABEE modified [β-D-Glcp-(1-4)]4-D-Glc. Experimental step for linkage 2 analysis is [487(2-) 652 341 161 fragments], (a) MS2 beam-type CID: [487(2-) fragments], (b) MS3 ion trap CID: [487(2-) 652 fragments], (c) MS4 ion trap CID: [487(2-) 652 341 fragments], and (d) MS5 ion trap CID: [487(2-) 652 341 161 fragments]. Experimental step for linkage 3 analysis is [487(2-) 406(2-) 341 161 fragments], (e) MS3 ion trap CID: [487(2-) 406(2-) fragments], (f) MS4 ion trap CID: [487(2-) 406(2-) 341 fragments], and (g) MS5 ion trap CID: [487(2-) 406(2-) 341 161 fragments].
As mentioned above, dissociation patterns of disaccharide ions (m/z 341) in the
negative ion mode have been used to assign linkages using sector instruments, triple
quadrupoles, Fourier transform ion cyclotrons and ion traps.7-11,13,24 Disaccharides having
2- or 6-linkages can be readily assigned in ion traps directly by MS2 as they yield
relatively abundant product ions of m/z 221 and 263, or m/z 221, 251 and 281,
respectively. For example, MS3 CID of the disaccharide substructure (α-D-Glcp-(1-6)-D-
(a)
200 400 600 800m/z
487310
652 814341 406 5035450
50
100
Rel
. Int
. (%
)-2H2-
Y3, m/z 652Y4, m/z 406(2-)
487(2-)
487(2-)
406(2-) 406(2-)
341
161
487(2-)487(2-)
406(2-)
341
200 400 600m/z
652
310
161407250
341
(e)
0
50
100
Rel
. Int
. (%
)
100 180 260 340m/z
161
341
179 281
(f)
0
50
100
60 80 100 120 140 160m/z
83
101
16173 115 143133
(g)
9787 1250
50
100
200 400 600m/z
652
328341 472 490310 606
(b)
0
50
100
Rel
. Int
. (%
)
100 180 260 340m/z
161
341
179
(c)
0
50
100
60 80 100 120 140 160m/z
83101
73133115 1619787 143125113
(d)
0
50
100487(2-)
652
487(2-)
652
341
652
341
161
487(2-)
C3C2
Y3Y42- Y4
[β-D-Glcp-(1-4)]4-D-Glc-ABEE, m/z 487(2-)
The parent ion The product ion for CID in the next stage
103
Glc, m/z 341) from α-D-Glcp-(1-6)-α-D-Glcp-(1-4)-D-Glc-18O (Figure 4.9b) produced
abundant ions of m/z 221, 251, and 281, characteristic of the 1-6 linkage. However,
especially for 3-linkages and to some extent for 4 linkages, depending on the specific
sugars involved, the A ions derived by dissociation processes occurring within the
reducing sugar can be relatively low in abundance (frequently less than 5% of the base
product peak) with very abundant product ions observed at m/z 161 and 179 due to
cleavage on either side of the glycosidic oxygen. In these cases, further
isolation/dissociation of the m/z 161 ions are highly preferred specifically because, under
defined dissociation conditions, they are nearly entirely comprised of the diagnostic Z1
isomers derived from the reducing sugar (Fig. 4.6b and c). For example, MS3 CID of the
disaccharide substructure (α-D-Galp-(1-3)-D-Gal, m/z 341) derived from α-D-Galp-(1-3)-
β-D-Galp-(1-4)-D-Gal-18O (Figure 4.10b) only revealed one observable cross-ring
fragment at m/z 281 (signature fragments of the 1-3 linkage: m/z 251 and 281 but no
263), making it difficult to assign the linkage position. Thus for 3- and 4-linked
disaccharides, the CID data of their Z1 (m/z 161) ions are particularly useful. Another
key advantage is that the reducing sugar of an oligosaccharide can be selectively 18O-
labeled, whereby the Z1 product ion obtained directly from the reducing sugar (m/z 163)
can be used to determine the reducing-end linkage, without requiring prior isolation of a
reducing disaccharide fragment. In some cases this is very valuable because the reducing
disaccharide may only be produced as a trace product ion or not, apparently, at all.
104
4.3.4 Linkage Determination for a Branched Oligosaccharide
In earlier discussions, we have shown that the Z1 ion approach can be applied to
linear oligosaccharides. However, in a real situation, branched oligosaccharides are often
encountered, such as N-linked glycans. We explored a way to convert a branched
oligosaccharide into linear structure using exoglycosidases and applied Z1 ion approach
for the smaller linear structures. Branched mannose 5 (Man5, having linkages of α1-3
and α1-6, structure shown in Scheme 4.2) is one of the common core structure in N-
glycans and was chosen as a model to test this method. First step is the chromophore
attachment to the reducing end to enable detection during later separation processes.
Schiff base formation with ABEE (without reduction) was employed for the initial tests.
Modified Man5 was purified by HPLC to eliminate substantial amount of impurities (i.e.
smaller oligosaccharides and isomers from the supply of commercial sample). ABEE
modified Man5 eluted around 10 min as shown in the chromatogram, Figure 4.13a.
There are two branching points within Man5 and they need to be cleaved to produce a
linear structure. An exoglycosidase, α1-2,3 Mannosidase, which cleaves off α1-2 and α1-
3 bonds was used to convert Man5 into a linear structure as shown in step 3. After
purification (step 4), a linear trisaccharide, α-D-Manp-(1-6)-α-D-Manp-(1-6)-D-Man-M,
was obtained. This ABEE modified Man3 came out around 7 min in Figure 4.13b. The
two HPLC separations (steps 2 and 4) are very important to eliminate possible impurities
and extract only the digested products originally from Man5. Step 5 is the delabeling of
chromophore and step 6 is 18O-labeling.
105
Scheme 4.2 Sample preparation steps for branched oligosaccharide before MS analysis.
M
α1-2,3 Mannosidase digestion
Delabeling (M)
HPLC separation
MS analysis
Reducing end labeling (M)
HPLC separation
M
M
Step 1
Step 2
Step 3
Step 4
Step 5
Step 7
18O-labelingStep 618O
106
A set of MS3 and MS4 experiments was required to obtain information for
linkages 1 and 2 within 18O-labeled α-D-Manp-(1-6)-α-D-Manp-(1-6)-D-Man:
([505 163 fragments] and [505 341 161 fragments]). Z1 and C2 ions were
formed under MS2 beam-type CID (CE = 20 V) as shown in Figure 4.14a. MS3 CID (ion
trap, 10 mVpp) of the Z1 ion clearly showed the distinct fragmentation pattern of a 1-6
linkage position (Figure 4.14c). MS3 CID (ion trap, 5 mVpp) of C2 ions (m/z 341) (Figure
4.14b) produced Z1 ions (m/z 161). MS4 CID (ion trap, 20 mVpp) of the Z1 ions (m/z 161)
is shown in Figure 4.14d. The fragmentation pattern of Figure 4.14c and d matched well
to the 1-6 linkage standard spectra (Figure 4.3d and 4.7d, respectively) and allowed
confident assignment of this linkage position.
The total amount of Man5 used for this experiment was only 1.2 nmol which is
compatible to permethylation method. Permethylation has the advantage of obtaining the
linkage information from the whole structure, it might experience the miss-assignment of
linkage positions due to ion suppression. In that case, Z1 method with a combination of
exoglycosidase can be used as a complimentary method to obtain confident linkage
information.
107
Figure 4.13 HPLC chromatograms from (a) step 2 and (b) step 4 separations.
0
400
800
1200
1600
2000
0 3 6 9 12
mAU
Retention time (min)
0
20
40
60
80
100
120
140
0 3 6 9 12
mAU
Retention time (min)
Man5-M
Man3-M
(a) Step 2 HPLC sepration
(b) Step 4 HPLC sepration
108
Figure 4.14 MSn CID spectra for the determination of linkage types within trisaccharides digested from Man5, α-D-Manp-(1-6)-α-D-Manp-(1-6)-D-Man-18O: (a) MS2 beam-type CID: [505 fragments], (b) MS3 ion trap CID: [505 341 fragments], (c) MS3 ion trap CID: [505 163 fragments], (d) MS4 ion trap CID: [505 341 161 fragments].
(b)
60 140 220 300m/z
0
50
100
0
50
100
Rel
. Int
. (%
)
(a)
100 200 300 400 500m/z
505
(c)505
163
505
341
505
341
161
(d)
60 80 100 120 140 160m/z
C2
α-D-Manp-(1-6)-α-D-Manp-(1-6)-D-Man-18O, m/z 505
60 80 100 120 140 160m/z
0
50
100
Rel
. Int
. (%
)R
el. I
nt. (
%)
383
341 443 505179 281 413
161 163
Z1
341
281
179
323221 251161
101
163103 135133
101
161131
109
4.3.5 Algorithm-Assisted Linkage Determination
For the possibility of automation of the linkage analysis in future developments, it
is desirable to use an algorithm for spectral-matching. Spectral similarity scores can be
calculated between MSn CID spectra of Z1 ions from oligosaccharides following selected
dissociation pathways and the averaged CID spectra of Z1 ions from the disaccharide
standards originating from 2-, 3-, 4-, or 6-linkages. The same equation introduced in
Chapter 2 (Eq. 2.1) was used.
For disaccharide standards, two sets of standard spectra were prepared. One was
based on the averaged CID of m/z 163 spectra as shown in Figure 4.3 and the other was
generated based on the averaged CID of m/z 161 spectra formed under ion trap CID with
low energy from native glucose-dimers shown in Figure 4.7. The spectral similarity score
for CID of m/z 163 from unknowns was calculated against CID of m/z 163 from
standards while the spectral similarity score for CID of m/z 161 from unknowns was
calculated against CID of m/z 161 from standards. Table 4.2 summarizes the similarity
scores of MSn CID spectra of Z1 ions for each linkage derived from the three
oligosaccharides studied. The highest spectral similarity scores always corresponded to
the correct linkage types with the values very close to unity. Note that the similarity
scores for incorrect linkage assignments were typically smaller than 0.8.
110
Table 4.2 Spectral similarity scores of MSn CID of Z1 ion spectra derived from oligosaccharides vs. disaccharide standards.
Oligosaccharides
Linkage (from
reducing-end)
Disaccharide Standards
1-2 1-3 1-4 1-6
α-D-Glcp-(1-6)-α-D-Glcp-(1-4)-D-Glc
1 0.7583 0.7053 0.9917 0.4566
2 0.8668 0.5212 0.7971 0.9944α-D-Galp-(1-3)-β-D-Galp-(1-4)-D-Gal
1 0.7603 0.7045 0.9912 0.4180
2 0.7223 0.9937 0.4805 0.4848
[β-D-Glcp-(1-4)]4-D-Glc
1 0.7722 0.6979 0.9702 0.5169
2 0.7504 0.4878 0.9917 0.7907
3 0.7619 0.5230 0.9875 0.7670
4 0.7511 0.5253 0.9938 0.7762α-D-Manp-(1-6)-α-D-Manp-(1-6)-D-Man
1 0.7807 0.5702 0.6247 0.9561
2 0.8907 0.5850 0.7598 0.9770
111
4.4 Conclusions
A new approach for linkage determination of oligosaccharides has been evaluated
based on MSn (n = 3-5) CID of Z1 ions. MS3 CID of 18O-labeled disaccharides
(deprotonated ions) enabled the discovery of Z1 ions as “diagnostic ions” for linkage
determination. The fragmentation pattern of Z1 ions was only sensitive to their linkage
positions and not to their sugar identities and anomeric configurations. This unique
property allowed standard CID spectra of Z1 ions to be generated from a small set of
disaccharides (possibly from 4 disaccharides) that were representative of many other
possible isomeric structures. The formation of Z1 ions could be optimized using ion trap
CID at lower activation energies vs. their structural isomers. This enabled their analysis
to be performed on native disaccharides or disaccharide subunits formed by MS/MS of
larger oligosaccharides. This information can be used in conjunction with that from the
m/z 341 disaccharide ion dissociation patterns to provide highly confident assignments of
all linkages. It is worthy of note that for 3- and 4-linked disaccharides, information from
the Z1 ions of unlabeled disaccharides is especially valuable for their linkage assignments
as they highly predominate over other isomers under low or high energy dissociation
conditions. With 2- and 6-linked disaccharides, lower energy ion-trap conditions are
preferable to yield a preponderant Z1 ion. While a small amount of an isomeric species
cannot be eliminated, fragmentation patterns of the ions isolated under optimal conditions
still enabled the linkage to be assigned. Yet the 2- and 6-linkages yield distinct
fragmentation patterns directly from dissociation of their disaccharide precursors with
abundant and characteristic sets of product ions,5-9,28 so for a completely unknown,
unlabeled disaccharide, this information would be highly useful in conjunction with
112
information from Z1 ion dissociation should there be any suspicion as to linkage
assignment. As we have not analyzed all stereochemical variants of Z1 product ions
arising from all linkages, it seems reasonable to surmise that higher statistical variance of
the linkage-characteristic dissociation patterns shown in Figures 4.3 and 4.7 may be
encountered, although observations so far indicate that linkage isomers dissociate with
dramatic differences. MSn CID of Z1 ions was applied to two trisaccharides and one
pentasaccharide to assess whether their linkage positions could be determined. By
comparing the MSn CID spectra of Z1 ions derived from different locations within
oligosaccharides with the standard CID spectra of Z1 ions from disaccharides, confident
assignments for individual linkages together with their locations within the oligomers
could be achieved for all three oligosaccharides studied as model compounds. This
method was further applied to a branched oligosaccharide, Man5. An exoglycosidase (α1-
2,3 Mannosidase) converted Man5 into a linear trisaccharide having the structure of α-D-
Manp-(1-6)-α-D-Manp-(1-6)-D-Man and two linkage information were successfully
obtained. The process of comparison of the Z1 ion spectra to standards was greatly
enhanced by a spectral similarity score algorithm, which provided numeric values in the
range of 0-1 with the highest scores indicative of the most likely assignment for the
linkage position. Although the examples were demonstrated on a hybrid triple
quadrupole/linear ion trap mass spectrometer, the MSn CID approach should in principle
be possible to implement on any standalone ion trap instrument, depending on the relative
abundances of isolated product ions derived from different oligosaccharide structures.
113
4.5 References
(1) Domon, B.; Müller, D. R.; Richter, W. J., Org. Mass Spectrom. 1989, 24, 357-359.
(2) Spengler, B.; Dolce, J. W.; Cotter, R. J., Anal. Chem. 1990, 62, 1731-1737.
(3) Zhou, Z.; Ogden, S.; Leary, J. A., J. Org. Chem. 1990, 55, 5444-5446.
(4) Hofmeister, G. E.; Zhou, Z.; Leary, J. A., J. Am. Chem. Soc. 1991, 113, 5964-5970.
(5) Dongre, A. R.; Wysocki, V. H., Org. Mass Spectrom. 1994, 29, 700-702.
(6) Ashline, D.; Singh, S.; Hanneman, A.; Reinhold, V., Anal. Chem. 2005, 77, 6250-6262.
(7) Ballistreri, A.; Montaudo, G.; Garozzo, D.; Giuffrida, M.; Impallomeni, G.; Daolio, S., Rapid Commun. Mass Spectrom. 1989, 3, 302-304.
(8) Garozzo, D.; Giuffrida, M.; Impallomeni, G.; Ballistreri, A.; Montaudo, G., Anal. Chem. 1990, 62, 279-286.
(9) Dallinga, J. W.; Heerma, W., Biol. Mass Spectrom. 1991, 20, 215-231.
(10) Carroll, J. A.; Willard, D.; Lebrilla, C. B., Anal. Chim. Acta 1995, 307, 431-447.
(11) Mulroney, B.; Traeger, J. C.; Stone, B. A., J. Mass Spectrom. 1995, 30, 1277-1283.
(12) Guan, B.; Cole, R. B., J. Am. Soc. Mass Spectrom. 2008, 19, 1119-1131.
(13) Fang, T. T.; Zirrolli, J.; Bendiak, B., Carbohydr. Res. 2007, 342, 217-235.
(14) Guan, B.; Cole, R. B., Rapid Commun. Mass Spectrom. 2007, 21, 3165-3168.
(15) Carroll, J. A.; Ngoka, L.; Beggs, C. G.; Lebrilla, C. B., Anal. Chem. 1993, 65, 1582-1587.
(16) Reinhold, V. N.; Reinhold, B. B.; Costello, C. E., Anal. Chem. 1995, 67, 1772-1784.
(17) Chen, S.-T.; Her, G.-R., J. Am. Soc. Mass Spectrom. 2012, 1-11.
(18) Xie, Y.; Lebrilla, C. B., Anal. Chem. 2003, 75, 1590-1598.
(19) Adamson, J. T.; Håkansson, K., Anal. Chem. 2007, 79, 2901-2910.
(20) Zhao, C.; Xie, B.; Chan, S.-Y.; Costello, C.; O’Connor, P., J. Am. Soc. Mass Spectrom. 2008, 19, 138-150.
114
(21) Wolff, J.; Laremore, T.; Aslam, H.; Linhardt, R.; Amster, I. J., J. Am. Soc. Mass Spectrom. 2008, 19, 1449-1458.
(22) Wolff, J.; Amster, I. J.; Chi, L.; Linhardt, R., J. Am. Soc. Mass Spectrom. 2007, 18, 234-244.
(23) Yu, X.; Huang, Y.; Lin, C.; Costello, C. E., Anal. Chem. 2012. 84, 7487-7494.
(24) Fang, T. T.; Bendiak, B., J. Am. Chem. Soc. 2007, 129, 9721-9736.
(25) Harvey, D. J., J. Am. Soc. Mass Spectrom. 2000, 11, 900-915.
(26) Chiesa, C.; Horváth, C., 1993, 645, 337-352.
(27) Konda, C.; Bendiak, B.; Xia, Y., J. Am. Soc. Mass Spectrom. 2012, 23, 347-358.
(28) Sheeley, D. M.; Reinhold, V. N., Anal. Chem. 1998, 70, 3053-3059.
115
CHAPTER 5 THE EFFECTS OF REDUCING END MODIFICATION ON GAS-PHASE CEHMISTRY OF SMALL OLIGOSACCHARIDES
5.1 Introduction
Derivatization of sugars such as permethylation1,2 of hydroxyl groups and
reducing end modification3,4 is widely applied strategy in structural analysis of
carbohydrates prior to mass spectrometric analysis to achieve improved sensitivity. These
derivatizations largely affect gas-phase dissociation chemistry of glycan ions.5,6 One
popular approach for reducing end derivatization is amination (formation of Schiff base)
between an aromatic amine (e.g., 2-aminobenzamide,7 4-aminobenzoic acid ethyl ester8)
and a reducing sugar due to the high reaction efficiency and simple one-pot procedure.
Reductive amination is the reduced form of amination and the reducing sugar stays in
open-ring form.9,10 Her et al. previously reported that not only the type of derivatives but
also the structure of reducing sugar ring (open or closed) affected the fragmentation
patterns. They reported that closed-ring structure enhanced cross-ring cleavages as
compared to open-ring form.11
MSn (n>2) approach has been demonstrated on a modified triple-
quadrupole/linear ion trap instrument for stereo-structure characterization of individual
sugar units within small linear oligosaccharides in Chapter 3. This approach (as shown in
Scheme 3.1) involves optimizing the formation of an ordered set of overlapping
116
disaccharide. Subsequently, the diagnostic product ion (m/z 221) having the structure of
non-reducing sugar glycosidically linked to a glycolaldehyde formed by cross-ring
cleavages were obtained. The success of this MSn approach largely depends on the
formation of the correct ladder of precursor ions within the tree of analysis, including Ym,
C2, and m/z 221 ions from C2. Stereochemistry and anomeric configuration of 3 sugar
units (3, 4, and 5 counting from the reducing-end sugar) in ABA modified (by reductive
amination) pentasaccharide anomeric isomers were successfully obtained. However,
sugar unit 2 information, which was theoretically possible to obtain by this approach, was
unavailable due to the absence of diagnostic m/z 221 ions from CID of Y2 ions.
Herein, a couple of N-derivatives (ABA, ABEE) were applied to model
disaccharides (α- and β-D-Glcp-(1-4)-D-Glc) by amination and reductive amination and
their fragmentation behavior, especially for the formation of m/z 221 ion, was studied.
Reducing-end derivatized disaccharides are considered to have the same structure with
the Y2 ions from oligosaccharides and can be used to simulate the condition without prior
MS/MS steps. Amination or reductive amination of N-derivatization showed a huge
difference on their fragmentation. Reductive N-derivatization produced no m/z 221 ions
while N-derivatization without reduction produced m/z 221 ions. This phenomenon is
consistent with that m/z 221 ion formation (from cross-ring cleavages) is favored by
having the reducing sugar in a closed-ring structure. N-derivatization without reduction
was then applied to trisaccharides. However CID of the deprotonated ion did not
produce Y2 ions. Since N-derivatization could not satisfy both criteria, we tested a new
derivatization: O-derivatization. O-derivatization gives the closed-ring structure of the
reducing-end sugar unit and the formation of m/z 221 ions was expected. To our
117
knowledge, there is no systematic studies on the fragmentation behavior caused by
different types of derivatives and also this is the first time to introduce O-derivatized
sugars for mass spectrometric analysis. Among five derivatives (HBA, HBEE, HBSA,
1,2HNA, and 3,2HNA), HBA and HBSA showed a formation of m/z 221 ions and were
applied to trisaccharides to confirm whether they form Y2 ions or not. HBSA showed the
formation of Y2 ions seemed successful, however, this derivative failed when linkage
isomers of α- and β-D-Glcp-(1-4)-D-Glc were used.
5.2 Experimental
5.2.1 Materials
Eight types of reducing end modifications (shown in the table 1 with estimated
pKa values) were performed on a series of disaccharides and trisaccharides to study their
effect on the gas-phase fragmentation of oligosaccharide anions. β-D-Glcp-(1-4)-D-Glc,
α-D-Glcp-(1-6)-α-D-Glcp-(1-4)-D-Glc, [α-D-Glcp-(1-4)]2-D-Glc, and [β-D-Glcp-(1-4)]4-
D-Glc, H218O, chloroform, sodium cyanoborohydride, acetic acid, dimethyl sulfoxide
(DMSO), acetic anhydride, dichloromethane (DCM), boron trifluoride diethyl etherate
(BF3·Et2O), sodium methoxide were purchased from Sigma-Aldrich, Inc. (St. Louis, MO)
Octaacetyl α-D-Glcp-(1-4)-β-D-Glc from Carbosynth, Ltd. (Berkshire, UK). Detailed
procedures of 18O-labeling,12 reductive amination (R)3,13, and amination without
reduction (NR) of reducing saccharides were described in the previous chapters (18O-
labeling in chapter 2 and reductive amination in chapter 3, and amination without
reduction in chapter 4).
118
Table 5.1 List of reducing end derivatives
.*Superscript e beside pKa value indicates estimation.
5.2.2 O-Derivatization at Reducing Sugars
Overall reaction steps are illustrated in Scheme 3.1 and (1) through (3) in
Scheme3.1 correspond to the each following section.
5.2.2.1 Acetylation of hydroxyl groups on sugars
Disaccharides or oligosaccharides (30 nmol) were dissolved in 60 to 100 μl of
acetic anhydride with 0.5 to 1 mg of iodine in an eppendorf tube and the reaction was
conducted with a stirring magnet for 3 h. The progress of the reaction was monitored by
MS for each step. After the completion of acetylation of hydroxyl groups on sugars,
DCM and aqueous sodium thiosulphate solution were added to the reaction mixture and
mixed thoroughly. The colorless organic layer was transferred to another eppendorf tube
and was washed with aqueous sodium carbonate solution to neutrality. The organic layer
was again transferred and dried completely by Speed-Vac.
CoreStructure R1 R2 (pKa of R2) Chemical Name (Abbreviation)
NH2COOH (4.87e)
COOCH2CH3 (-)4-aminobenzoic acid (ABA)
Ethyl-4-aminobenzoate (ABEE)
OHCOOH (4.57)
COOCH2CH3 (-)SO3H (2.54)
4-hydroxybenzoic acid (HBA)Ethyl 4-hydroxybenzoate (HBEE)
4-hydroxybenzenesulfonic acid (HBSA)
OH COOH (3.28e) 1-hydroxy-2-napthoic acid (1,2HNA)
OH COOH (3.02e) 3-hydroxy-2-napthoic acid (3,2HNA)
119
5.2.2.2 O-derivatization to the acetylated sugars14
1 mg of acetylated sugar, 1.5 mg of derivatives (HBA, HBEE, HBSA, 1,2HNA,
or 3,2HNA), 1 μL of BF3·Et2O were dissolved in 500 μL of DCM under nitrogen. The
reaction mixture was placed in ultrasonic bath (B3500A-MT, VWR, Radnor, PA) for 1 to
2 h. After the completion of O-derivatization, aqueous sodium carbonate solution was
added and mixed thoroughly. The organic layer was transferred and dried completely by
Speed-Vac.
5.2.2.3 Deacetylation of O-derivatized Acetylated Sugars
1 mg of O-derivatized acetylated sugars and 1mg of sodium methoxide were
dissolved in 500 μL of methanol and incubated at 60 oC for 10-24 h.
5.2.2.4 Desalting of O-derivatized Sugars by HPLC
The reaction mixture of deacetylated O-derivatized sugars was desalted using
Agilent 1200 series HPLC system (Agilent Technologies, Santa Clara, CA). Desalting
was carried out on a HILIC (PolyLC Inc., Columbia, MD) column at a flow rate of 0.4
mL/min with a linear gradient of 20-35% solvent A in 30 min. Solvent A was water and
solvent B was acetonitrile. The eluent was detected at a different wavelength suitable for
individual chromophores (HBA: 310 nm, HBEE: 252 nm, HBSA: 271 nm, 1,2HNA: 352
nm, 3,2HNA: 352 nm).
120
Scheme 5.1 Reaction steps for O-derivatization at reducing sugars
5.2.3 Mass Spectrometry
All samples were analyzed in the negative ion mode using a 4000Qtrap mass
spectrometer (Applied Biosystems/Sciex, Toronto, Canada) equipped with a home-built
nanoelectrospray (nanoESI) source. Two types of low energy collisional activation
methods were accessible on this instrument, i.e. beam-type CID and ion trap CID.
Analyst 1.5 software was used for instrument control, data acquisition, and processing.
The typical parameters of the mass spectrometer used in this study were set as follows:
spray voltage, -1.1 to -1.5 kV; curtain gas, 10; declustering potential, 50 V; beam-type
CID collision energy (CE), 5 to 30 V; ion trap CID activation energy (AF2), 5 to 60
(arbitrary units); scan rate, 1000 m/z/s; pressure in Q2, 5.0 x 10-3 Torr, and in Q3, 2.5 x
10-5 Torr.
121
5.3 Results and Discussion
In order to investigate the major fragments formed from a native disaccharide,
MS2 beam-type and ion trap CID spectra of 18O-labeled β-D-Glcp-(1-4)-D-Glc were
collected and shown in Figure 5.1a. Product ions observed were m/z 161 (B1, -182 Da),
163 (Z1, -180 Da), 179 (C1, -164 Da), 221 (A2, -122 Da), 263 (A2, -78 Da), 283 (A2, -60
Da) and 325 (-18 Da). Product ions at m/z 161, 163, and 179 are formed from glycosidic
bond cleavages and m/z 221, 263, and 283 are formed from cross-ring cleavages. The
m/z 221 ion is the diagnostic ion for stereo-structure analysis and can be observed from
any disaccharides (although abundance may vary), however, Y1 ion (m/z 181) was never
observed in the product ions from CID of native disaccharide anions.
5.3.1 Gas-Phase Fragmentation Studies of N-Derivatized Disaccharides
MS2 beam-type and ion trap CID spectra of ABA derivatized disaccharide by
amination (β-D-Glcp-(1-4)-D-Glc-NR-ABA, MW: 461 Da) are shown in Figure 5.1b.
The major fragments included peaks at m/z 161 (B1), 178 (X0), 179 (C1), 192 (X0), 220
(X0), 221 (A2), 234 (X0), 262 (X0), 263 (A0), and 280 (Z1). The relatively intensity of m/z
161, 179, 221, 263 ions were similar to the β-D-Glcp-(1-4)-D-Glc-18O (Figure 5.1a),
however, a variety of X0 ions (cross-ring cleavages consisting of the reducing end) were
produced abundantly which suggested that the charge stayed on reducing end due to the
presence of carboxylic acid group in the derivative. As previously reported, beam-type
CID produced more fragments from cross-ring cleavages (A and X ions, especially m/z
221 ion) which required higher activation energies, while glycosidic bond cleavage (Z1
122
ion formation) was enhanced by on-resonance ion trap CID (slow heating of the ion
which favors the lowest energy fragmentation channels).15
MS2 beam-type and ion trap CID spectra of ABA derivatized disaccharide by
reductive amination (β-D-Glcp-(1-4)-D-Glc-R-ABA, MW: 463) are shown in Figure
5.1c. These spectra showed major peaks at m/z 161 (B1), 179 (C1), 204 (X0), 222 (X0),
234 (X0), 246 (X0), 264 (X0), 282 (Z1), 300 (Y1), and 402 (X1). Unlike non-reduced
derivatization (Figure 5.1b), Y1 ion was formed, however, there were no A ions such as
m/z 221, 263, and 281. Similar phenomena were observed from the pare of β-D-Glcp-(1-
4)-D-Glc-NR-ABEE (MW: 489) vs. β-D-Glcp-(1-4)-D-Glc-R-ABEE (MW: 491) as
shown in Figure 5.1d and e, respectively. The A type ions were only observed from β-D-
Glcp-(1-4)-D-Glc-NR-ABEE while Y1 ion was only observed from β-D-Glcp-(1-4)-D-
Glc-R-ABEE. Comparing ABA and ABEE derivatives (e.g., Figure 5.1b and d, A, B,
and C type ions consisting of the non-reducing end were always observed in higher
intensities in ABEE than ABA which is consistent with the lower acidity of ABEE
derivative than ABA.
123
Figure 5.1 MS2 beam-type vs. ion trap CIDs from (a) β-D-Glcp-(1-4)-D-Glc-18O, (b) β-D-Glcp-(1-4)-D-Glc-NR-ABA, (c) β-D-Glcp-(1-4)-D-Glc-R-ABA, (d) β-D-Glcp-(1-4)-D-Glc-NR-ABEE, and (e) β-D-Glcp-(1-4)-D-Glc-R-ABEE.
Z
Ion trap
X
Ion trap
282
222204 462300
179246
234
264C
Z
Y1X
282
462
222
179 300
246
402264
204161 CB
Z
Y1
X
Ion trap
248308
179 263290
488221161281
B
CZ
A
A
A
X
X
248
263 308179
488281220290161
ZC
B
A
AX
X
X
Ion trap310
179 490430328161
250 400Y1
Z
CBX
XX
310328
232250
179 490283
164
161
Z
Y1
CB XX
Ion trap
163
179343263283145
161
221AAA
CB
Z
163
343179325263 283
AAC
Z
Beam type
Beam type
Beam type Beam type
(a) 18O
343
(b) NR-ABA
460
(c) R-ABA
462
(d) NR-ABEE
488
(e) R-ABEE
490
100 200 300 400 5000
50
100R
el. I
nt. (
%)
280
263220
179 460442161192
Z
262C
B
XA
X 281A
161B
A221
100 200 300 400 5000
50
100
Rel
. Int
. (%
)
Beam type
B
C
220 280
179 262460
221
161 234192
178
A
263A
X
X
X
100 200 300 400 5000
50
100
Rel
. Int
. (%
)
100 200 300 400 5000
50
100
Rel
. Int
. (%
)
100 200 300 400 5000
50
100
234
100 200 300 400 5000
50
100
Rel
. Int
. (%
)
100 200 300 400 5000
50
100
Rel
. Int
. (%
)
164
100 200 300 400 5000
50
100
100 200 300 400 5000
50
100
100 200 300 400 5000
50
100
221A
X
X
X
X
m/z m/z
161
179 163
221
283
161
179 280
221 161
179 282
161
179 308
221
281
300
161
179 310
328
124
5.3.2 Gas-Phase Fragmentation Studies of O-Derivatized Disaccharides
MS2 beam-type and ion trap CID spectra of β-D-Glcp-(1-4)-D-Glc-HBEE (MW:
489), β-D-Glcp-(1-4)-D-Glc-HBA (MW: 461), and β-D-Glcp-(1-4)-D-Glc-HBSA (MW:
497) are shown in Figure 5.2a, b, and c, respectively. β-D-Glcp-(1-4)-D-Glc-HBEE
showed major peaks at m/z 165 (Y0), 323 (B2), and 411 (X1) and small amount of m/z 221
ion was formed by beam-type CID (Figure 5.2a). α-D-Glcp-(1-4)-D-Glc-HBA (Figure
5.2b) showed major peaks at m/z 137 (Y0), 263 (A2), and 323 (B2). α-D-Glcp-(1-4)-D-
Glc-HBSA (Figure 5.2c) showed major peaks at m/z 173 (Y0), and 335 (Y1) while small
amount of m/z 221 (A2) ion was formed by both beam-type and ion trap CID. HBEE and
HBA derivatives showed abundant glycosidic bond cleavages between a reducing end
sugar and a derivative, forming Y0 and B2 ions depending on the charge locations. The
ratio of these two ions changes depending on the type of CID used. Bean-type CID
produced Y0 ions as the base peak while ion trap CID produced B2 ions as the base peak.
Based on these results, the majority of the original deprotonation sites can be considered
as located at the derivatized function groups and beam-type CID (fast reaction, < 1 ms)
produces abundant Y0 ions. The deprotonation sites are moved down to the sugar side
while ions were staying in the trap by ion trap CID (slow reaction, > 100 ms). HBSA
showed a little different effect on the formation of Y0 and B0 ions. Beam-type CID
produced 100% of Y0 ion (base peak) and about 40% of Y1 ion while no B2 ion was
formed. Ion trap CID reduced the formation of Y0 ion and increased the formation of Y1
ion, however, again, no B2 ion was generated. This is probably due to the higher acidity
of HBSA (pKa = 2.54) as compared to HBEE and HBA (pKa = 4.57) and the negative
charge tends to be localized on sulfonic acid group.
125
MS2 beam-type and ion trap CID spectra of β-D-Glcp-(1-4)-D-Glc-1,2HNA
(MW: 511) and β-D-Glcp-(1-4)-D-Glc-3,2HNA (MW: 511) are shown in Figure 5.2d and
e, respectively. Both medication groups showed very similar fragments, having a base
peak at m/z 229 (X0), and fragments at m/z 187 (Y0), 313 (X0), and 331 (Z1). All of the
fragmentation appeared were X, Y, Z type of ions and no A, B, C ions were formed. This
is also due to the higher acidity of HNA (pKa=3.02 – 3.28) as compared to HBEE and
HBA (pKa = 4.57).
126
Figure 5.2 MS2 beam-type vs. ion trap CID from (a) β-D-Glcp-(1-4)-D-Glc-HBEE, (b)β-D-Glcp-(1-4)-D-Glc-HBA, (c) β-D-Glcp-(1-4)-D-Glc-HBSA, (d) β-D-Glcp-(1-4)-D-Glc-1,2HNA, and (e) β-D-Glcp-(1-4)-D-Glc-3,2HNA.
100 200 300 400 5000
50
100R
el. I
nt. (
%)
100 200 300 400 5000
50
100
Rel
. Int
. (%
)165
323 489161221245178 411
B2BA A
Y0
X
323
411 489435165 244 453287
B2
Y0
X
Ion trap
Beam type
100 200 300 400 5000
50
100
Rel
. Int
. (%
)
100 200 300 400 5000
50
100
Rel
. Int
. (%
)
(a) HBEE
(b) HBA
(c) HBSA
100 200 300 400 5000
50
100
Rel
. Int
. (%
)
100 200 300 400 5000
50
100
Rel
. Int
. (%
)
100 200 300 400 5000
50
100
100 200 300 400 5000
50
100
100 200 300 400 5000
50
100
100 200 300 400 5000
50
100
(e) 3,2HNA
(d) 1,2HNA
Ion trap
Beam type
Ion trap
Beam type
137
323 461263245
B2A
Y0
323
461263137 245
B2
AY0
173
335497255
283 317
241
215
172Z
Y1
X
Y0
335
173
497417281215
221437
Y1
A
A XX
Y0
489
461
497
511
511
229
187 511313 331 391
ZX X
X
Y0
229
511187
361 375
157
443
313 467347 493
185
X
X
XY0
229157
185187 511479143167 473313
295
271
331493ZY0
X
XX
229
157 511187
331
313
493295
349271
483
185
Z
Y1Y0
XX
X
Ion trap
Beam type
Ion trap
Beam type X
X
m/z
m/z
161
179
221
323
165
161
179 323
137
335173
317
221
281
187331
187
331
A
A
127
5.3.3 Comparisons between N-Derivatized (by Amination) and O-Derivatized Trisaccharides
Non-reduced form of ABA and ABEE, HBA and HBSA derivatized disaccharides
produced m/z 221 ions (diagnostic ions for stereo-chemical information) and these
derivatives are thus applied to trisaccharides to test whether Y2 ions can be formed or not.
The formation of Y2 ions is the key to obtain sugar 2 information from above listed
derivatized oligosaccharides because the MSn CID steps needed to follow for sugar 2 are:
[M-H]- Y2 221 fragments). MS2 beam-type and ion trap CID spectra of α-D-Glcp-
(1-6)-α-D-Glcp-(1-4)-Glc-NR-ABA and α-D-Glcp-(1-6)-α-D-Glcp-(1-4)-D-Glc-NR-
ABEE are shown Figure 5.3a and b, respectively. α-D-Glcp-(1-6)-α-D-Glcp-(1-4)-D-
Glc-NR-ABA showed major peaks at m/z 161 (B1), 179 (C1), 192 (X0), 220 (X0), 221
(A2), 262 (X0), 280 (Z1), 323 (A2), 341 (C2), 383 (A3), 425 (A3), and 562 (X2). α-D-Glcp-
(1-6)-α-D-Glcp-(1-4)-D-Glc-NR-ABEE showed very similar fragmentation patterns,
having different masses for X, Y, Z ions due to the different masses in derivatives.
However, no Y2 ions were produced from CID of α-D-Glcp-(1-6)-α-D-Glcp-(1-4)-D-
Glc-ABA and α-D-Glcp-(1-6)-α-D-Glcp-(1-4)-D-Glc-ABEE and these derivatizations
were not applicable to our MSn (n>2) approach.
O-derivatized trisaccharides were then examined to see the formation of Y2 ions
from MS2 CID. MS2 beam-type and ion trap CID spectra of [α-D-Glcp-(1-4)]2-D-Glc-
HBEE and [α-D-Glcp-(1-4)]2-D-Glc-HBSA are shown Figure 5.4b and c, respectively.
Spectra from [α-D-Glcp-(1-4)]2-D-Glc-HBEE showed a base peak at m/z 485 (B3) and
small amount of product ions at m/z 165(Y0), 323 (B2), 383 (A3), 425 (A3), and 573(X2).
As was previously observed in α-D-Glcp-(1-4)-D-Glc-HBEE, glycosidic bond cleavages
128
between a reducing end sugar and a derivative was the main fragmentation site. α-D-
Glcp-(1-4)-D-Glc-HBEE showed different base peaks (Y0 for beam-type and B2 for ion
trap), there was no significant difference between beam-type and ion trap CIDs for [α-D-
Glcp-(1-4)]2-D-Glc-HBEE. Spectra from [α-D-Glcp-(1-4)]2-D-Glc-HBSA showed major
peaks at m/z 173 (Y0), 335, (Y1), 497 (Y2) with small amount of product ions at m/z 215
(X0), 221 (A2), 317 (Z2), 383 (A3), 479 (Z2), and 581 (A3). HBSA derivatized
trisaccharide produced a variety of product ions, most importantly having Y2 ion and m/z
221 ions.
Figure 5.3 MS2 beam-type CID from (a) α-D-Glcp-(1-6)-α-D-Glcp-(1-4)-D-Glc-NR-ABA, and (b) α-D-Glcp-(1-6)-α-D-Glcp-(1-4)-D-Glc-NR-ABEE.
0
50
100
Rel
. Int
. (%
)
100 200 300 400 500 600 700
0
50
100
Rel
. Int
. (%
)
100 200 300 400 500 600 700
383
220 622
341280
179
425
323
221161
562
C1
B1X2
A2
A2
C2
Z1
192 X0
248
650383308
341220
179 425
321
323
C1
A2
A2
C2
Z1
(a) NR-ABA
(b) NR-ABEE
622
650
m/z
221A2
Beam type
Beam type
A2
A2
X0
B2
161179
221323
341280
161179
221323
341308
B1161
323B2
m/z
B2
129
Figure 5.4 MS2 beam-type vs. ion trap CID from (a) [α-D-Glcp-(1-4)]2-D-Glc-18O, (b)[α-D-Glcp-(1-4)]2-D-Glc-HEE, (c) [α-D-Glcp-(1-4)]2-D-Glc-HBA, and (d) [α-D-Glcp-(1-4)]2-D-Glc-HBSA.
100 200 300 400 5000
50
100
Rel
. Int
. (%
)
161341
425383179
505443281263221 323
163
B1
C1
A2 B2
C2Z1
100 200 300 400 5000
50
100
Rel
. Int
. (%
)
341
443 505161425179 281 487
163
C2
Z1
C1
B1
A2A2
100 200 300 400 500 6000
50
100 383
425
137 407485 623545221
179323161
311
587281
100 200 300 400 500 6000
50
100 485
545407
425383 623
605
587323
341305
461281
501221
381
B1
C1
Y2
C2
B3
B3 X2
Y0
B2
B2
100 200 300 400 500 6000
50
100
Rel
. Int
. (%
)
485
651383 512425 623573323165
100 200 300 400 500 600m/z0
50
100
Rel
. Int
. (%
)
485
651573425383 512 623
B3
B3
B2 X2
X2
A3Y0A3
100 200 300 400 500 6000
50
100 173
497335659
215 317 419 581221 479383
100 200 300 400 500 6000
50
100497
335 659215 383281
Y2
Y2
Y1
Y1
Z2Z1
Y0
X0
X0
X1
505 623
651
(c) HBA
(d) HBSA
659
(a) 18O
(b) HBEE
Ion trap
Beam type
Ion trap
Beam type
Ion trap
Beam type
Ion trap
Beam type
m/z
A3A3
A3
A3A3
A2
A2A2/Z2
A2
A3
A3
X2A3A2 A2
A3A3
A3
A2 A3
A2 A3
X2
161179
323
341163
161179
323
341281
137
323
165485 317173
335
479
497
130
The relative intensity (normalized to the base peak) of the specific product ions
formed by different types of derivatizations was summarized in Table 5.2. Specific
product ions of our interest were m/z 221 ions from disaccharide-derivatives and Y2 ions
from trisaccharide-derivatives. Both criteria were only satisfied by HBSA. HBSA was
then derivatized to disaccharide linkage isomers (e.g., α-D-Glcp-(1-2)-D-Glc, α-D-Glcp-
(1-3)-D-Glc, β-D-Glcp-(1-3)-D-Glc, α-D-Glcp-(1-6)-D-Glc, and β-D-Glcp-(1-6)-D-Glc)
to test the applicability of this derivatives to a variety of disaccharides. Unfortunately,
m/z 221 ion could not be obtained from linkage isomers listed above.
Table 5.2 Summary of the normalized product ion intensity of characteristic ions by a variety of derivatizations
Relative intensity of product ions (%)
Type of derivativesDisaccharide Trisaccharide
m/z 221 ion Y2 ion
Native 5 0NR-ABA 15 0R-ABA 0 -
NR-ABEE 14 0R-ABEE 0 -
HBA 0 4HBEE 3 0HBSA 2 100
1,2HNA 0-
3,2HNA 0
131
5.4 Conclusions
The fragmentation behavior of oligosaccharides can be affected by the chemical
nature of reducing-end derivatizations. HBSA was the only derivative which produced
both Y2 and m/z 221 ions from a derivatized model trisaccharide, [α-D-Glcp-(1-4)]2-D-
Glc-HBSA. However, this derivative only worked for 1-4 linked sugars and could not be
applied for other linkage isomers.
We continue to explore different derivatives, especially having the pKa value of
between 3.5 and 4.5 would be a good choice since HBSA (pKa = 2.54) was too acidic
and produced mainly Y ions while HBA (pKa = 4.57) was not acidic enough to produce
Y ions.
132
5.5 References
(1) Ciucanu, I.; Kerek, F., Carbohydr. Res. 1984, 131, 209-217.
(2) Mechref, Y.; Kang, P.; Novotny, M. V., Solid-Phase Permethylation for Glycomic Analysis. In Glycomics, Packer, N.; Karlsson, N., Eds. Humana Press: 2009; Vol. 534, pp 53-64.
(3) Harvey, D. J., J. Am. Soc. Mass Spectrom. 2000, 11, 900-915.
(4) Harvey, D. J., J. Chromatogr. B 2011, 879, 1196-1225.
(5) Cancilla, M. T.; Penn, S. G.; Carroll, J. A.; Lebrilla, C. B., J. Am. Chem. Soc. 1996,118, 6736-6745.
(6) Orlando, R.; Allen Bush, C.; Fenselau, C., Biol. Mass Spectrom. 1990, 19, 747-754.
(7) Ruhaak, L. R.; Huhn, C. H.; Deelder, A. M.; Wuhrer, M., Anal. Chem. 2008, 80,6119-6126.
(8) Cheng, H.; Pai, P.; Her, G.-R., 2007, 18, 248-259.
(9) Her, G. R.; Santikarn, S.; Reinhold, V. N.; Williams, J. C., 1987, 6, 129-139.
(10) Nishikaze, T.; Kaneshiro, K.; Kawabata, S.; Tanaka, K., Anal. Chem. 2012, 84,9453-9461.
(11) Chen, S.-T.; Her, G.-R., J. Am. Soc. Mass Spectrom. 2012, 23, 1408-1418.
(12) Fang, T. T.; Bendiak, B., J. Am. Chem. Soc. 2007, 129, 9721-9736.
(13) Chiesa, C.; Horváth, C., 1993, 645, 337-352.
(14) Smits, E.; Engberts, J. B. F. N.; Kellogg, R. M.; van Doren, H. A., J. Chem. Soc. Perkin Trans. 1 1996, 0, 2873-2877.
(15) Zaia, J., Mass Spectrom. Rev. 2004, 23, 161-227.
133
VITA
Chiharu Konda was born on December 22, 1979, in Nagano, Japan. She is the
daughter of Yoshie and Hiromich Konda. While enrolling at Meiji Gakuin
Higashimurayama High School (Tokyo, Japan), she transferred to Notre Dame College
School (Ontario, Canada) and graduated from both high schools in 1998. Chiharu started
working as a sales engineer at one of the largest cell phone companies in Japan, NTT
Docomo in 1999. After working for 6 years, she pursued her dream to study abroad for
higher education and enrolled at Valdosta State University (Georgia, USA) in 2005. She
worked in Professor De La Garza’s lab from 2006-2008, focusing on the fabrication of
TiO2 film by electro-deposition. In August of 2009, Chiharu graduated with a Bachelor of
Science in Chemistry with Magna Cum Laude and began her graduate career in the
Chemistry Department at Purdue Univeristy. She joined Professor Yu Xia’s lab in the fall
of 2009, where her research focused on structural analysis of carbohydrates using mass
spectrometry. She defended her Ph. D. thesis in October 2013. Following graduate
school, she will be working at the new division of NTT Docomo where developing the
synergetic technology involving analytical science and cell phone network.
134
PUBLICATIONS
1. Du, Y. M.; Konda, C.; Xia, Y.; Ouyang, Z. “Statistical Analysis Model for Classifying Stereo-Structures of Oligosaccharides Using Tandem Mass Spectrometry”, InPreparation.
2. Konda, C.; Bendiak, B.; Londry, F. A.; Xia, Y. “Stereochemistry and AnomericConfiguration with Single-Sugar Resolution for Oligosaccharides via MSn (n>2)”, InPreparation.
3. Konda, C.; Bendiak, B.; Xia, Y. “Linkage Determination of Linear Oligosaccharides by MSn (n>2) Collision-Induced Dissociation (CID) of Z1 Ions in Negative Ion Mode Tandem Mass Spectrometry”, J. Am. Soc. Mass Spectrom., accepted.
4. Konda, C.; Bendiak, B.; Xia, Y. “Differentiation of the Stereochemistry and Anomeric Configuration for 1-3 linked Disaccharides via Tandem Mass Spectrometry and 18O-Labeling”. J. Am. Soc. Mass Spectrom., 2012, 23, 347-358.
B American Society for Mass Spectrometry, 2011DOI: 10.1007/s13361-011-0287-5J. Am. Soc. Mass Spectrom. (2012) 23:347Y358
RESEARCH ARTICLE
Differentiation of the Stereochemistryand Anomeric Configuration for 1-3 LinkedDisaccharides Via Tandem Mass Spectrometryand 18O-labeling
Chiharu Konda,1 Brad Bendiak,2 Yu Xia1
1Department of Chemistry, Purdue University, West Lafayette, IN 47907-1393, USA2Department of Cell and Developmental Biology, and Program in Structural Biology and Biophysics, University of ColoradoDenver, Anschutz Medical Campus, Aurora, CO, USA
AbstractCollision-induced dissociation (CID) of deprotonated hexose-containing disaccharides (m/z 341)with 1–2, 1–4, and 1–6 linkages yields product ions at m/z 221, which have been identified asglycosyl-glycolaldehyde anions. From disaccharides with these linkages, CID of m/z 221 ionsproduces distinct fragmentation patterns that enable the stereochemistries and anomeric config-urations of the non-reducing sugar units to be determined. However, only trace quantities ofm/z 221ions can be generated for 1–3 linkages in Paul or linear ion traps, preventing further CID analysis.Here we demonstrate that high intensities of m/z 221 ions can be built up in the linear ion trap (Q3)from beam-type CID of a series of 1–3 linked disaccharides conducted on a triple quadrupole/linearion trap mass spectrometer. 18O-labeling at the carbonyl position of the reducing sugar allowedmass-discrimination of the “sidedness” of dissociation events to either side of the glycosidic linkage.Under relatively low energy beam-type CID and ion trap CID, anm/z 223 product ion containing 18Opredominated. It was a structural isomer that fragmented quite differently than the glycosyl-glycolaldehydes and did not provide structural information about the non-reducing sugar. Underhigher collision energy beam-type CID conditions, the formation of m/z 221 ions, which have theglycosyl-glycolaldehyde structures, were favored. Characteristic fragmentation patterns wereobserved for each m/z 221 ion from higher energy beam-type CID of 1–3 linked disaccharidesand the stereochemistry of the non-reducing sugar, together with the anomeric configuration, weresuccessfully identified both with and without 18O-labeling of the reducing sugar carbonyl group.
Key words: Oligosaccharides, Stereochemistry, Anomeric configuration, Collision-induceddissociation, Tandem mass spectrometry
Introduction
Carbohydrates play important roles in biological systems,such as providing energy to cells and functioning as
structural components for plant cell walls. By conjugatingwith proteins and lipids, carbohydrates are widely involvedin cell–cell interactions, cell signaling, and self and non-selfrecognition events [1, 2]. Carbohydrates can form almostunlimited variations in their structures due to their structuralcomplexity. In order to elucidate the structure of anoligosaccharide, it is necessary to characterize the stereo-chemistry of each monosaccharide unit, the anomericconfiguration of the glycosidic bonds, linkage positions,Received: 23 August 2011Revised: 20 October 2011Accepted: 21 October 2011Published nline: 18 November 2011
Electronic supplementary material The online version of this article(doi:10.1007/s13361-011-0287-5) contains supplementary material, whichis available to authorized users.
Correspondence to: Yu Xia; e-mail: [email protected]
o
135
and the sequence of the individual monosaccharides inthe oligomer. When enough sample is available, higher-dimensional NMR is a powerful tool to obtain detailedstructural information [3–5].
Mass spectrometry is a widely applied method instructural analysis of carbohydrates, due to its capabilityof providing detailed molecular information and highsensitivity [6]. The molecular weight information ofcarbohydrates is readily obtained from soft ionizationmethods such as electrospray ionization (ESI) [7, 8] andmatrix-assisted laser desorption/ionization (MALDI) [9–11]. Tandem mass spectrometry based on collision-induced dissociation (CID) is heavily relied on to obtainstructural information for carbohydrates. Glycosidic bondcleavages and/or cross-ring cleavages are typically ob-served from collisional activation. Following the nomen-clature proposed by Domon and Costello , A, B, and Cions are fragments containing the non-reducing terminuswhile X, Y, and Z ions include the reducing end [12].Four types of fragments, B, C, Y, and Z ions, areformed from cleavages on either side of the glycosidicoxygen. B and Y ions are cleaved at the non-reducingside of a glycosidic oxygen and C and Z ions arecleaved at the reducing side of a glycosidic oxygen.Based on the specific mass differences of fragmentsresulting from glycosidic bond cleavages, sequenceinformation for both linear and branching oligosacchar-ides as well as glycotypes such as the complex, hybrid,or high-mannose can be determined for methylatedoligosaccharides [8, 13].
A and X ions result from cross-ring cleavages and theyare typically more informative for the structural analysis ofcarbohydrates. It has been shown that the relative abundan-ces of these ions can be correlated to the linkage position,and in some cases, the anomeric configuration and stereo-chemistry of each monosaccharide. For example, CIDspectra of deprotonated di- and oligosaccharide alkoxyanions in the negative ion mode showed distinguishablefragmentation patterns for each linkage position, which wassuccessfully applied to di-, tri-, and hexasaccharides [14–17]. Negative ion adducts [18, 19] and positive adducts [20,21] of deprotonated di- and oligosaccharides have also
enabled linkage positions to be determined, both in thenegative and positive ion modes, and linkage sites can beestablished for neutral disaccharides as positive ion adductsof lithium or sodium ions or as protonated adducts [22–27].Determination of anomeric configuration has been demon-strated for underivatized disaccharides in the negative ionmode [15, 17, 18], for derivatized 1-4- and 1-6-linkeddisaccharides [28], and in the positive ion mode with alkalimetals [22–27] and lead cationization [20]. Prior knowledgesuch as linkage position, ring form, and stereochemistry [15,17, 18, 20, 22–28], however, was typically required toassign anomeric configuration, which was difficult to applyto larger oligosaccharide systems [18]. CID of metalcationized monosaccharides derivatized as a Schiff basewith diethylenetriamine [29] and CID of metal cationized N-acetylhexosamine diastereomers [30] have been shown toproduce distinct fragmentation patterns according to thestereochemistry. The assignment of stereochemistry has alsobeen obtained by matching the CID spectrum of acetylatedmonosaccharides in oligosaccharides with reference spectra[31, 32]. Using additional statistical analysis, anomericconfiguration has been identified for metal cationizedglucopyranosyl-glucose disaccharides (1–2, –3, –4, and –6linkages) [26]. Also, the linkage positions and stereo-chemistries of non-reducing units can be discriminated forglucose, galactose, and mannose containing disaccharideshaving 1–2, –3, and –4 linkages [27].
Recently, a tandem mass spectrometry approach has beendeveloped to differentiate the stereochemistry and anomericconfiguration for the non-reducing unit of hexose-containingdisaccharides having any of the 16 possible stereochemicalvariants [33, 34]. In this method, diagnostic ions at m/z 221were formed from CID of deprotonated disaccharide ions(m/z 341). It was established that the m/z 221 ions consistedof the intact non-reducing sugar glycosidically linked toglycolaldehyde, as indicated in Scheme 1 (where GAabbreviates glycolaldehyde). Note that an open-chain formfor the reducing sugar is indicated in Scheme 1 and also forother disaccharides discussed later. This is based on theobservation of absorbance in the carbonyl stretch region invariable wavelength infrared radiation photo-dissociation ofdeprotonated monosaccharide anions in the gas phase [35].
H --HH
-H --HCID CIDCID CIDCID CID
D Gl (1 4) Gl / 341 D Gl GA / 221α-D-Glcp-(1-4)-Glc m/z 341 α-D-Glcp-GA m/z 221α D Glcp (1 4) Glc, m/z 341 α D Glcp GA, m/z 221
Scheme 1. Formation of glycosyl-GA anions at m/z 221 from CID of deprotonated 1–4 linked disaccharides
348 C. Konda et al.: Structure Determination for Disaccharides
136
When m/z 221 ions were further dissociated by collisionalactivation, disaccharides having different non-reducing sugarunits and anomeric configurations showed distinct fragmen-tation patterns that matched synthetic glycosyl-GAs. Thismethod was shown to be useful for assigning the stereo-chemistry as well as the anomeric configuration of theglycosidic bond for the non-reducing sugar in disaccharideshaving 1–2, 1–4, and 1–6 linkages [33, 34]. However, due tothe low abundance of m/z 221 ions produced from 1–3linked disaccharides, MS3 CID of m/z 221 ions could not beperformed, and it was unclear whether the fragmentationpatterns could be used for assigning either their stereochem-istry or anomeric configuration.
Herein, a series of 1–3 linked disaccharides were studiedon a triple quadrupole-linear ion trap mass spectrometer(QTRAP 4000). MS3 CID data of m/z 221 ions from the 1–3linked disaccharides were obtained for the first time. Theformation of m/z 221 ions was examined using differentcollisional activation methods, i.e., beam-type CID and iontrap CID of the deprotonated disaccharides. 18O-labeling ofthe reducing carbonyl oxygen in 1–3 linked disaccharideswas used to enable mass-discrimination of structural isomersof the (usually) m/z 221 ions. By choosing the proper CIDconditions, the diagnostic m/z 221 ions (the glycosyl-GAs)could be formed as the dominant isomer. Their CIDfragmentation patterns could be used to establish thestereochemistry and anomeric configuration of the non-reducing sugar unit from 1–3 linked disaccharides.
ExperimentalMaterials
α-D-Glcp-(1–2)-D-Glc (kojibiose), β-D-Glcp-(1–2)-D-Glc(sophorose), α-D-Glcp-(1–3)-D-Glc (nigerose), α-D-Glcp-(1–3)-D-Fru (turanose), and H2
18O were purchased fromSigma-Aldrich, Inc. (St. Louis, MO, USA); β-D-Glcp-(1–3)-D-Glc (laminaribiose), α-D-Manp-(1–3)-D-Man (3α-manno-biose), and α-D-Galp-(1–3)-D-Gal (3α-galactobiose) werepurchased from Carbosynth, Ltd. (Berkshire, UK). α- and β-monosaccharide-glycolaldehyde standards, glucopyranosyl-glycolaldehydes (Glcp-GA), galactopyranosyl-glycolaldehydes(Galp-GA), and mannopyranosyl-glycolaldehydes (Manp-GA) were synthesized as previously described [33]. Dis-accharides and synthetic standards were dissolved in meth-anol to a final concentration of 0.01 mg/mL and NH4OHwas added to a final concentration of 1% immediately beforeuse.
18O-Labeling of Reducing Disaccharides
Disaccharides (1 mg) were dissolved in 100 μL of H218O for
3 to 10 d at room temperature. The solution was furtherdiluted to 0.1 mg/mL with methanol before mass spectro-metric analysis.
Mass Spectrometry
All samples were analyzed in the negative-ion mode on aQTRAP 4000 mass spectrometer (Applied Biosystems/SCIEX, Toronto, Canada) equipped with a home-builtnanoelectrospray ionization (nanoESI) source. A schematicpresentation of the instrument ion optics is shown in theSupporting Information, Figure S1. Two types of low energycollisional activation methods were accessible on thisinstrument, i.e., beam-type CID and ion trap CID. Inbeam-type CID, the precursor ions (m/z 341 or 343) wereisolated in Q1, accelerated in the Q2 collision cell forcollisional activation, and all products were analyzed in theQ3 linear ion trap. Collision energy (CE) was defined by thepotential difference (absolute value) between Q0 and Q2. Inion trap CID, the precursor ions were isolated in the Q3linear ion trap via the RF/DC mode and a dipolar excitationwas used for collisional activation. In order to perform iontrap CID at different Mathieu q-parameters, an AC (alter-nating current) generated from an external waveformgenerator (Agilent Technologies, Santa Clara, CA, USA)was used for resonance excitation. Frequency and the lowmass cut-off were calculated by SxStability (Pan GalacticScientific, Omemee, Ontario, Canada). MS3 CID experi-ments were carried out by first performing beam-type CIDof precursor ions in Q2. The fragment ions of interest wereisolated in Q3 and then subjected to ion trap CID. Analyst1.5 software was used for instrument control, data acquisi-tion, and processing. The typical parameters of the massspectrometer used in this study were set as follows: sprayvoltage, –1.1 to −1.5 kV; curtain gas, 10; declusteringpotential, 50 V; beam-type CID collision energy (CE), 5 to30 V; ion trap CID activation energy (AF2), 5 to 60(arbitrary units); scan rate, 1000 m/z; pressure in Q2, 5.0×10–3 Torr, and in Q3, 2.5×10–5 Torr. Ion injection time wascontrolled to keep a similar parent ion intensity: typically 3×106 counts per second (cps) for MS2 CID experiments and1×106 cps for MS3 CID experiments. Activation time waskept constant at 200 ms for all ion trap CID experiments.Seven spectra were collected for CID of m/z 221 ions fromsynthesized monosaccharide-GA standards (deprotonatedmolecules) and disaccharides over a 1 y period. Standarddeviations of peak heights were calculated for major frag-ments such as m/z 87, 99, 101, 113, 129, 131, 159, 161, 203,and 221, which were observed from all the standards anddisaccharides studied here except β-D-Glcp-GA and β-D-Glcp-(1–2)-D-Glc, which showed no peaks at m/z 99.
Results and DiscussionIon trap CID of deprotonated 1–3 linked disaccharides (m/z341) typically generates ions at m/z 221 in trace abundanceon a Paul trap instrument, and isolation or further CID of m/z221 ions have not been achieved before [14, 15, 34, 36]. The4000QTRAP mass spectrometer used in this study has aunique triple quadrupole-linear ion trap configuration,
C. Konda et al.: Structure Determination for Disaccharides 349
137
offering high sensitivity due to the large capacity of thelinear ion trap, and allowing either beam-type or ion trapcollisional activation. In beam-type CID, the precursor ionswere isolated in Q1 and accelerated in Q2 for collisionalactivation, while ion trap CID was conducted in Q3 with adipolar excitation for collisional activation. Since CIDfragmentation patterns can be sensitive to the means ofactivation, the formation of m/z 221 ions from five 1–3linked disaccharides was investigated via both beam-typeand ion trap CID. Figure 1 compares the MS2 beam-type andion trap CID of deprotonated β-D-Glcp-(1–3)-D-Glc (m/z341) using low energy CID conditions. A relatively low CE(6 V) was used for beam-type CID; in ion trap CID, the AF2for an AC dipolar excitation was set to 25 (arbitrary units)for 200 ms. Under either activation condition, the absolute
intensities of m/z 221 ions (indicated by an arrow inFigure 1) were very low and their relative intensities wereless than 1% (normalized to the base peak in the spectrum).This phenomenon was generally observed for all 1–3 linkeddisaccharides studied herein. The insets in Figure 1 demon-strate the isolated m/z 221 ions (with a 2 m/z isolationwindow) from each set of dissociation conditions. For beam-type CID, 1×106 cps of m/z 221 ions could be accumulatedwith an injection time of 1 s, which was sufficient forperforming the next stage of tandem mass spectrometry(MS3 in this case) with reasonable ion statistics andsensitivity. Far lower abundance of the m/z 221 ions (4.6×104 cps) could be isolated from ion trap CID of m/z 341,even after doubling the injection time to 2 s. As a result, itwas not feasible to obtain MS3 CID for m/z 221 ionsgenerated from m/z 341 precursor ions initially isolatedwithin the trap. In experiments described below, beam-typeCID was used to dissociate disaccharide precursor anions inthe Q2 collision cell thereby generating m/z 221 product ionsin high enough abundance to acquire their spectra in thelinear trap reproducibly.
CID of m/z 221 Ions Generated from 1–3 LinkedDisaccharides
Previous studies have demonstrated that m/z 221 productions formed from collisional activation of disaccharideanions typically consist of an intact non-reducing sugar witha 2-carbon aglycone derived from the reducing sugar [34].Three dominant fragment peaks are commonly observedfrom CID of m/z 221: m/z 101, 131, and 161. The relativeintensities of these peaks, together with some other fragmentions, can be used to establish the fragmentation patterns andto distinguish the stereochemistry and anomeric configura-tion of the non-reducing sugar. Given that the CID patternsof m/z 221 ions will be used for structural identification,spectral reproducibility is an important issue. Similar to thefindings from a Paul trap instrument [33, 34], we noticedthat the number of ions (m/z 221) in the linear ion trap andthe energy input into an ion were among the most importantparameters affecting spectral reproducibility. To ensurereasonable ion statistics and avoid adverse space chargeeffects, the intensity of the m/z 221 ions was kept at 1×106 cps before MS3 CID. Based on previous studies, the CIDenergies were tuned so that the ratio of remaining precursorion to the most abundant product ion was kept around 18%±3% [33]. Figure 2a, b, e, and f were the averaged spectrafrom seven repetitions collected over a 1 y period, and theywere further used to make spectral comparisons in laterdiscussion. Error bars in the spectra indicate the standarddeviation of the peak intensity for 10 major fragment ions,which were frequently observed for all the disaccharidesstudied herein (m/z 87, 99, 101, 113, 129, 131, 159, 161,203, and 221). The standard deviations for these peaks wereless than 5% in most cases, indicating high reproducibility ofthe spectra from day to day by controlling the ion counts in
341100 341
( ) 221100(a) 1.05e6
2211.05e6
ps
% cp
y, % y,
sity
sity
ns ns
ten
te
Int
222In
e I 222
ive
at 161
Rel
161179
R
113113143143
100 140 180 220 260 300 3400
60 100 140 180 220 260 300 340m/z
060
m/z
179100 179100161(b) 4 6e4 221161 4.6e4 221
s
341% cps
341
y,%
y, c
ty ity
nsi nsi
en en
nte nte
e In In
veat
iv
113ela
113143
Re
143
R
060 100 140 180 220 260 300 340
060
Figure 1. MS2 CID spectra in the negative ion modeobtained from deprotonated β-D-Glcp-(1–3)-D-Glc (m/z 341)under low energy dissociation conditions: (a) beam-type CID(CE=6 V), and (b) ion trap CID (AF2=25). Insets in (a) and (b)show the isolation of m/z 221 ions generated from beam-typeCID (injection time = 1 s) and ion trap CID (injection time = 2 s),respectively
350 C. Konda et al.: Structure Determination for Disaccharides
138
the trap before CID and the energy input to the ions. Sincethe CID patterns upon dissociation of m/z 221 ions can differto some extent from instrument to instrument [37], CIDspectra of the synthetic monosaccharide-GA were collectedas standards for comparisons. Figure 2a and b show the CIDdata of α- and β-D-Glcp-GA, respectively. The abundantpeaks at m/z 101 and 131 in Figure 2a are a signature of anon-reducing glucose with an α anomeric configuration.Note that a distinct fragmentation pattern is observed for theβ configuration (Figure 2b), where m/z 131 and 161 ions aredominant. The same collisional activation conditions wereapplied to m/z 221 ions derived from α-D-Glcp-(1–3)-D-Glcand β-D-Glcp-(1–3)-D-Glc, anomeric isomers containing anon-reducing glucose. It is obvious that the spectra from thetwo anomeric isomers (Figure 2c and d) were drasticallydifferent from their corresponding D-Glcp-GA standards,however, were similar to each other. This indicates that them/z 221 ions generated using low collision energies from m/z341 precursors have different structures from the D-Glcp-GAstandards, and that their CID patterns cannot be used to assign
either the stereochemistry or anomeric configurations of theions. Note that beam-type CID was used to generate the m/z221 ions from disaccharides shown in Figure 2, a conditiondiffering from previous studies where ion trap CID had beenused [33]. This difference in activation could have contributedto the formation of structural isomers observed for the m/z 221product ions. In order to test this hypothesis, m/z 221 ionsof 1–2 linked disaccharides, α-D-Glcp-(1–2)-D-Glc and β-D-Glcp-(1–2)-D-Glc, were formed using similar beam-type CIDconditions and further subjected to MS3 CID (Figure 2eand f). Except for a larger fluctuation in peak intensityfor m/z 203, almost identical fragmentation patterns tothe standards were observed (compare Figure 2a to e andb to f), strongly indicating that the expected D-Glcp-GAstructures were formed. We further investigated a widevariety of disaccharides and found that the CID patternsof m/z 221 ions matched with their corresponding monosac-charide-GA standards with the exception of 1–3 linkeddisaccharides when low collision energy beam-type CIDconditions were used to dissociate the disaccharides.
-
60 100 140 180 220m/z m/z
161
203131113
221
-H
α-D-Glcp-(1-3)-Glc, m/z 341
(c)
-H -α-D-Glcp-(1-2)-Glc, m/z 341
m/z 221 m/z 221
0
100
Rel
ativ
e In
ten
sity
, %
60 100 140 180 2200
100
Rel
ativ
e In
ten
sity
, %
87
101
113
129
131
159161
203
221
99
(e)
-H -α-D-Glcp-GA, m/z 221
60 100 140 180 220m/z
0
100
Rel
ativ
e In
ten
sity
, %
87
99101
113
129
131
159161203
221
(a)
(b)
-H -β-D-Glcp-GA, m/z 221
β-D-Glcp-(1-3)-Glc, m/z 341-H -
m/z 221
060 100 140 180 220
m/z
100
Rel
ativ
e In
ten
sity
, %
161
203131113
221
(d)
60 100 140 180 220m/z
0
100
Rel
ativ
e In
ten
sity
, %
87101 113
129
131
159
161
203
221
--H
β-D-Glcp-(1-2)-Glc, m/z 341
m/z 221
60 100 140 180 220m/z
0
100
Rel
ativ
e In
ten
sity
, %
87101113
129159
161
221
131
(f)
Figure 2. MS2 ion trap CID spectra of m/z 221 ions derived from synthetic standards (a) α-D-Glcp-GA, AF2=25 and (b) β-D-Glcp-GA, AF2=18. MS3 CID spectra of m/z 221 ions derived from low energy beam-type CID of glucose-containingdisaccharides: (c) α-D-Glcp-(1–3)-Glc, CE=6 V for MS2 and AF2=25 for MS3, (d) β-D-Glcp-(1–3)-Glc, CE=6 V for MS2
and AF2=25 for MS3, (e) α-D-Glcp-(1–2)-Glc, CE=5 V for MS2, and AF2=27 for MS3, and (f) β-D-Glcp-(1–2)-Glc, CE=5 V for MS2, and AF2=25 for MS3. The error bars in the spectra show the standard deviation of the peak intensitybased on seven spectra collected over a 1 y period
C. Konda et al.: Structure Determination for Disaccharides 351
139
18O-labeling at the carbonyl position of the reducingsugar was used to mass-discriminate the “sidedness” ofdissociation events to either side of the glycosidic linkageand thus the origins of the m/z 221 and/or potential 223product ions. Figure 3 compares relatively low energy beam-type and ion trap CID of 18O-labeled deprotonated α-D-Glcp-(1–3)-D-Glc, m/z 343. Similar fragments were ob-served for both conditions; however, the ion abundance form/z 283 (loss of 60 Da, C2H4O2) was much higher in beam-type CID relative to ion trap CID. It is possible that thisfragmentation channel requires higher activation energy andis promoted, even in lower-energy beam-type CID, sincehigher collision energies (several eV) may have beenobtained as compared to ion trap CID (hundreds of meV).
The inset in Figure 3a shows data collected using a wideisolation window (6 m/z units) around m/z 221 after CID ofm/z 343 precursor ions. A peak at m/z 223, due toincorporation of 18O, appeared with much higher abundancethan m/z 221 ions for both low-energy beam-type and iontrap CID. Note that if the expected D-Glcp-GA structurewere formed, it should consist of the intact non-reducingsugar unit with a 2-carbon aglycon derived from thereducing sugar (C-2 and C-3 or C-3 and C-4). In this case,18O should not be incorporated into the product ion and itshould still appear at m/z 221. Therefore, the observationof abundant m/z 223 ions indicated that under relativelylow energy CID conditions, most of the m/z 221 ionsformed from α-D-Glcp-(1–3)-D-Glc do not have the D-Glcp-GA structure which is the structural isomer neededto distinguish the stereochemistry and anomeric config-uration of the non-reducing sugar.
Ions at m/z 221 and m/z 223 were further subjected to iontrap CID. Figure 4 compares the CID spectra of the isolatedm/z 221 ions and the m/z 223 ions generated from 18O-labeled α-D-Glcp-(1–3)-D-Glc and β-D-Glcp-(1–3)-D-Glc.The CID spectra of the m/z 221 ions (Figure 4a and c) fromthe two anomeric isomers are distinct from each other andalmost identical to those of the corresponding α and β-D-Glcp-GA standards (Figure 2a and b). The fragmentationpatterns of m/z 223 (Figure 4b and d), however, were similarto each other yet were very different from the syntheticglucosyl-GA standards.
The major fragments that resulted from CID of the m/z223 ions included product ions at m/z 205, 163, 131, and113. These ions are likely due to losses of water (−18 Da), a2-carbon piece, C2H4O2 (−60 Da), a 3-carbon pieceincluding 18O, C3H6O2+
18O (−92 Da), and sequential orconcerted losses of a three-carbon piece plus waterincluding 18O (−110 Da), respectively. Interestingly, neitherthe loss of water nor the loss of 60 Da significantly involvesloss of the 18O oxygen. The m/z 223 ions are hypothesizedto have a structure in which the reducing sugar is connectedto a two-carbon piece from the non-reducing sugar as shownin the scheme above Figure 4b and d. Note that C-1 is nolonger chiral on the piece from the (former) non-reducingsugar, which also explains the similarity in the CID data ofthe m/z 223 ion derived from the two anomeric isomers(Figure 4b and d). We also noticed some subtle differencesbetween Figure 4b and d. For example, the relativeintensities of m/z 205 and 159 are higher (more than 10%)in Figure 4b than in d. These differences may be due to theexistence of a small fraction of structural isomers other thanthat hypothesized for the m/z 223 ions.
The Effect of CID Conditions on the Formationof m/z 221/223 Product Ions from 3-LinkedDisaccharides and their 18O-Labeled Isotopomers
As demonstrated in Figure 4, abundant structural isomers ofm/z 221 product ions, (m/z 223 ions from the 18O-labeled
343100 343223100 (a)
(b)
2.9e5
s
% ps
y, % cp
ity
ty,
ns sit 221
ten
ens
nt te
eI In
ive
0
ati
2830
ela 283
Re
163179179
0100 140 180 220 260 300 340
060
m/z
343223100
4 0 4223
4.0e4
s
% ps
y, % c
sity ty,
ns sit
221ten
ens 221
Int
nte
e I In
ive
0at 0
Rel 179R 179163
100 140 180 220 260 300 340060 100 140 180 220 260 300 340
m/z
060
m/z
Figure 3. MS2 spectra of 18O-labeled α-D-Glcp-(1–3)-D-Glcunder (a) relatively low-energy beam-type CID (CE=6 V), and(b) ion trap CID (AF2=25). Insets in (a) and (b) show isolationof m/z 221 and 223 ions generated from beam-type CID(injection time = 1 s) and ion trap CID (injection time = 2 s),respectively
352 C. Konda et al.: Structure Determination for Disaccharides
140
disaccharides) were observed under relatively low-energydissociation conditions of 1–3 linked disaccharides, eitherusing beam-type or ion trap CID. This prevents theassignment of the stereochemistry or anomeric configurationof the non-reducing sugar in a typical scenario where either adisaccharide is unlabeled or when it is isolated (unlabeled)from a larger oligosaccharide structure. It would be highlydesirable to optimize CID conditions or, for that matter, tofind any dissociation conditions whereby the relatively pure,structurally informative glycosyl-GA (m/z 221) ions couldbe formed predominantly. Figure 5 shows the effect ofcollision energies on the formation of m/z 221 and 223 ionsunder beam-type and ion-trap CID, using 18O-labeled β-D-Glcp-(1–3)-D-Glc as an example. The data were collectedusing a wide isolation window around m/z 221 to observeboth m/z 221 and 223 ions.
It is clear from Figure 5a–c that the collision energy inbeam-type CID affects the absolute and relative intensities of
m/z 221 ions. When the CE was relatively low (CE=5 V), m/z223 ions were predominantly formed, with four times higherintensity than that of m/z 221. At a higher CE (CE=10 V), m/z221 and 223 ions were seen at nearly equal intensities. Once theCE was increased to 15 V, m/z 221 ions became the dominantpeak, accounting for 80% of the total intensities from m/z 221and 223. Further increasing CE, however, resulted in a hugeloss of ion abundance possibly due to competitive ion ejectionthus the ratio was not improved.
Parameters that might affect the formation of m/z 221 ionsversus m/z 223 ions were also examined for ion trap CID.When ion trap CID of m/z 343 was performed under theinstrument default Mathieu q-parameter (q=0.235), m/z 223ions were formed exclusively independent of activationenergies (data not shown). By changing the activation Mathieuq-parameter to a higher value, precursor ions are placed under ahigher potential well depth, and higher activation energies canbe applied. An AC generated from an external waveform
60 100 140 180 220m/z
0
100
Rel
ativ
e In
ten
sity
, %
131
101
99
22111387159
203
(b)(a)
-H - m/z 223 -H -
α-D-Glcp-(1-3)-Glc, m/z 343 m/z 221 -H
-H -
-α-D-Glcp-(1-3)-Glc, m/z 343
60 100 140 180 220m/z
163
205
113 131
223
0
100
Rel
ativ
e In
ten
sity
, %
159161
1818
(d)
-H - m/z 223 -H -
β-D-Glcp-(1-3)-Glc, m/z 343m/z 221 -H
-H --
β-D-Glcp-(1-3)-Glc, m/z 343
(c)
0
100
Rel
ativ
e In
ten
sity
, %
60 100 140 180 220m/z
0
100R
elat
ive
Inte
nsi
ty, %
163
113 205131
223159
60 100 140 180 220m/z
131
161
22187
203159101
18
18
Figure 4. MS3 spectra of m/z 221 and 223 ions derived from 18O-labeled α-D-Glcp-(1–3)-D-Glc and β-D-Glcp-(1–3)-D-Glc. (a)CID of m/z 221 ions, CE=15 V (MS2), AF2=15 (MS3), (b) CID of m/z 223 ions, CE=5 V (MS2), AF2=14 (MS3) from 18O-labeled α-D-Glcp-(1–3)-D-Glc, and (c) CID of m/z 221 ions, CE=15 V (MS2), AF2=28 (MS3), (d) CID of m/z 223 ions, CE=5 V (MS2), AF2=24 (MS3) from 18O-labeled β-D-Glcp-(1–3)-D-Glc
C. Konda et al.: Structure Determination for Disaccharides 353
141
generator was used for resonance excitation at q=0.4. Asshown in Figure 5d to f, the ratio ofm/z 221 tom/z 223 ions wasincreased from almost zero to about 1 as the activationamplitude was increased from 100 mVp-p to 400 mVp-p
(activation time: 50 ms for all cases). Further increasing theactivation amplitude resulted in a decrease in m/z 221 to 223ratio as well as a huge ion loss.
The data in Figure 5 suggest that m/z 221 ions, whichhave the desired monosaccharide-GA structures, are gener-ated more favorably under relatively high collision energyconditions in both beam-type and ion trap CID. Comparedwith ion trap CID, beam-type CID provided more abundantand higher relative intensities of the m/z 221 ions that werewanted for discrimination of the stereochemistry andanomeric configuration of the non-reducing sugar. Evidently,a higher activation energy is needed for the formation ofthese m/z 221 product ions, and the pathway to generate theglycosyl-GAs is favored when the internal energies of themolecular ions increase. In beam-type CID, much highercollision energies can be applied (typically more than 10 V)as compared to ion trap CID (hundreds of mV), which leadsto a shift in the internal energy distribution of the molecularions to the high energy direction [38]. It is interesting topoint out that the glycosyl-glycolaldehyde product ions arevirtually the only isomeric species generated under ion trapor low-energy beam-type CID of the 1–2 linked disaccharideanions [34]. Since much higher relative intensities of theglycosyl-glycolaldehyde product ions (10%–40%, normal-ized to the most abundant peak) can be formed from 1–2linkages compared with that of 1–3 linkages (typically G1%
relative intensity) under ion trap CID conditions, it isreasonable to conclude that the formation of these ions from1–2 linkages needs less energy than required for theirgeneration from 1–3 linkages. Therefore, the formation ofthe glycosyl-glycolaldehyde product ions is a much lowerenergy dissociation channel for 1–2 linked disaccharides buta fragmentation pathway for this isomeric species can onlybe promoted for 1–3 linked disaccharides when the collisionenergy is higher.
Given the high pressure in the collision cell (~5 mTorr),multiple collisions happen in beam-type CID, and the first-generation product ions may also be subjected to collisionalactivation once they are formed within the collision cellespecially under higher CE conditions. In this sense, beam-type CID is less selective than ion trap CID, where fragmentions are not typically further activated. Indeed, MS3 CIDstudies in the ion trap showed that many fragment ions,including m/z 325, 323, 283, 281, 253, and 251 generatedm/z 221 ions, which might contribute to the observation ofhigher intensity m/z 221 ions under beam-type CID due tosecondary dissociation.
Identification of the Non-Reducing Sugarand its Anomeric Configuration for 1–3 LinkedDisaccharides
Since relatively pure m/z 221 ions containing the intact non-reducing sugars could be formed using beam-type CID withhigh collision energies, it was possible to differentiate thestereochemistry and anomeric configuration of the non-
223 221 2213.9e5 223 2.5e5221
223 3.9e5 221( ) (b)
2.5e5 223 3.9e5(a) (b) (c)
s
(a) (b) (c)
s s
ps ps
cps
c c
y, c
ty,
ty,
ty,
sit
sit
nsi
ens
221 ens
en
nte 221
nte nte
In 223In In
0 0 0221 223 221 223 221 223
0 0 03
m/z221 223
m/z221 223
m/z
223 223 2239 1e4
2232 0e5
2231 0e5 223
2219.1e4 2.0e5 1.0e5 221(d) (e) (f)(d) (e) (f)
ps
ps
ps
cp cp cp
y,
y,
y,
221sity
sity
sity
221ns
ns
ns
ten
ten
ten
Int
Int
Int
I I
0 0 0221 223 221 223 221 2230 0 0221 223
m/z221 223
m/z221 223
m/zm/z m/z m/z
Figure 5. Isolation of m/z 221 and 223 ions derived from 18O-labeled β-D-Glcp-(1–3)-D-Glc under different collisionalactivation conditions. Beam-type CID: (a) CE=5 V, (b) CE=10 V, (c) CE=15 V. Ion trap CID at q=0.4, f=119.248 kHz, excitationtime = 50 ms: (d) 100 mVp-p, (e) 250 mVp-p, (f) 400 mVp-p
354 C. Konda et al.: Structure Determination for Disaccharides
142
reducing sugar in disaccharides without 18O-labeling. Figure 6shows the MS3 CID spectra of m/z 221 ions generated bybeam-type CID with relatively high CE (13 to 22 V) from five1–3 linked disaccharides. Each spectrum was an average ofseven spectra and the error bars indicate standard deviations ofthe peak intensities. The standard deviations were found to behigher (0%–12%) for the disaccharide samples than those fromstandards (0%–4%). This larger degree of spectral variation islikely contributed by the fluctuation in ion intensity of the lowabundance m/z 221 isomers under slightly different instrumentconditions, and these isomers fragment differently from thediagnostic and more abundant m/z 221 ions that have themonosaccharide-GA structures.
Note that α-D-Glcp-(1–3)-D-Glc, α-D-Glcp-(1–3)-D-Fru,and β-D-Glcp-(1–3)-Glc are disaccharide isomers containinga glucose as the non-reducing sugar; however, each haseither a different anomeric configuration or reducing sugar.The characteristic fragmentation profile for disaccharideshaving glucose as the non-reducing sugar and an α-anomeric
configuration can be clearly identified for Figure 6a (α-D-Glcp-(1–3)-D-Glc) and Figure 6c (α-D-Glcp-(1–3)-D-Fru),which is distinct from the β-anomeric isomer as shown inFigure 6b (β-D-Glcp-(1–3)-D-Glc, compare all three to thesynthetic standards shown in Figure 2a and b). Figure 6dshows the characteristic fragmentation profile for the m/z221 ion of disaccharides having galactose as the non-reducing sugar and having an α-anomeric configuration(compare to Figure S3a, CID of m/z 221 from α-D-Galp-GAstandard, Supporting Information). The MS3 CID of α-D-Manp-(1–3)-Man (Figure 6e) was similar to that of the α-D-Manp-GA (Supporting Information, Figure S3b). It is alsoimportant to note that under low-energy dissociation condi-tions, the spectra of the m/z 221 product ions derived from thedisaccharides α-D-Glcp-(1–3)-D-Fru, α-D-Galp-(1–3)-D-Galand α-D-Manp-(1–3)-Man did not match those of theirrespective glycosyl-glycolaldehydes (Supporting Informa-tion, Figure S2b and Figure S3c and d). We concludethat this is due to the presence of alternate isomers, pos<
(b)
-H -α-D-Glcp-(1-3)-Glc, m/z 341
m/z 221
β-D-Glcp-(1-3)-Glc, m/z 341-H -
m/z 221
60 100 140 180 220m/z
0
100
Rel
ativ
e In
ten
sity
, %
87
101113
129
131
159
161
203
221
60 100 140 180 220m/z
0
100
Rel
ativ
e In
ten
sity
, %
87
99101
113
129
131
159161
203
221
(a) (c)
-H -α-D-Glcp-(1-3)-Fru, m/z 341
m/z 221
60 100 140 180 220m/z
0
100
Rel
ativ
e In
ten
sity
, %
87
99
101
113
131
129 159
161
203
221
-H -α-D-Galp-(1-3)-Gal, m/z 341-H -
α-D-Manp-(1-3)-Man, m/z 341
m/z 221 m/z 221
(d)
60 100 140 180 220m/z
0
100
Rel
ativ
e In
ten
sity
, %
8799
101
113129131
159
161
203
221
60 100 140 180 220m/z
0
100
Rel
ativ
e In
ten
sity
, %
8799101
113
129
131
159
161
203221
(e)
Figure 6. MS3 CID of m/z 221 ions generated via using high CE (CE=13 to 22 V) for the dissociation of deprotonateddisaccharide ions (m/z 341). (a) α-D-Glcp-(1–3)-D-Glc, CE=15 V (MS2), AF2=26 (MS3), (b) β-D-Glcp-(1–3)-D-Glc, CE=13 V(MS2), AF2=30 (MS3), (c) α-D-Glcp-(1–3)-D-Fru, CE=18 V (MS2), AF2=25 (MS3), (d) α-D-Galp-(1–3)-D-Gal (3α-Gal-Gal),CE=22 V (MS2), AF2=35 (MS3), and (e) α-D-Manp-(1–3)-D-Man (3α-Man-Man), CE=20 V (MS2), AF2=36 (MS3). The error barsin the spectra show the standard deviation of peak intensities based on seven spectra collected over a one year period
C. Konda et al.: Structure Determination for Disaccharides 355
143
sibly related in their origins to the hypothetical structuresshown in Figure 4b and d but having different reducingmonosaccharides.
The methodology for assigning the stereochemistry andanomeric configuration for the non-reducing sugar unitwithin a disaccharide is based on the comparison of theCID patterns of m/z 221 ions to those of the syntheticmonosaccharide-GA standards [33]. A high similaritybetween the compared spectra indicates a large likelihoodof them sharing the same structure. Spectral similarityscores, which have been widely used in mass spectral librarysearch for both small molecules [39], peptides [40–42], andoligosaccharides [43], were chosen to facilitate thesecomparisons. The spectral similarity scores were calculatedbetween each of the averaged spectra in Figure 6 and theaveraged spectra from the monosaccharide-GA standardsbased on the following equation [44],
Spectral similarity score ¼P
kI1mI2m
� �1=2P kI1mþI2m
2
; and k¼P
I2mPI1m
where I1m and I2m are the normalized intensities of an ion atm/z = m for the two spectra. Note that the spectral similarityscore always has a value between 0 and 1. If two spectra areexactly the same, the spectral similarity score becomes 1. Ingeneral, a large similarity score indicates close similaritybetween the two spectra and a large degree of structuralsimilarity. As shown in Table 1, the spectral similarityscores between a standard and a disaccharide having thesame stereochemistry and anomeric configuration for thenon-reducing side were the highest scores, ranging between0.9838 and 0.9977. When a disaccharide’s stereochemistryand anomeric configuration on the non-reducing side did notmatch with the standard, the spectral similarity score wassignificantly lower, between 0.6942 and 0.9178. Clearly, bycomparing the spectral similarity scores, assigning thestereochemistry and anomeric configuration for the non-reducing side of the 1–3 linked disaccharides could beachieved with high confidence. Note that this was onlypossible under high-energy beam-type CID conditions wherethe m/z 221 product anions containing the intact non-reducing sugars were optimally generated from precursordisaccharides.
ConclusionsCollisional activation of deprotonated 1–3 linked hexose-containing disaccharides (m/z 341) generated a low-abundancem/z 221 product ion. By 18O-labeling the reducing sugarcarbonyl oxygen of these disaccharides, at least twostructural isomers of the m/z 221 ion with the main portionderived from either side of the glycosidic linkage could bemass-discriminated (m/z 221 vs. m/z 223), which enabledthe isomers to be isolated and independently studied. Them/z 221 isomer containing the intact non-reducing sugarattached in glycosidic linkage to a glycolaldehyde aglyconwas found to be analytically useful, since CID of thisspecies provided the structural information that identifiedthe stereochemistry and anomeric configuration of the non-reducing sugar. No structural information could be obtainedfrom m/z 223 isomer(s) to determine the stereochemistry ofthe non-reducing sugar or its anomeric configuration. Theformation of the diagnostic m/z 221 isomer was found tobe affected by CID conditions and was favored underhigher energy beam-type CID. It was demonstrated thatunder optimized CID conditions, this structural isomercould be generated predominantly from five different 1–3linked disaccharides without requiring 18O-labeling of thereducing sugar. Identification of the non-reducing sugar andthe anomeric configuration, therefore, were achieved at ahigh confidence level by statistically comparing the CIDdata of m/z 221 ions generated from the disaccharidesamples with those of the synthetic standards via spectralsimilarity scores. This study also demonstrated that beam-type CID was a more desirable activation method comparedwith ion trap CID to characterize disaccharides using themethodology based on the CID patterns of m/z 221 ions.This method now enables the anomeric configuration andstereochemistry of the m/z 221 ions derived from 1–2, –3, –4,or –6 linked disaccharides to be assigned in the negative ionmode. This capability was afforded due to the specificarrangement of the triple quadrupole-linear ion trap instru-ment. It combined (1) selection of the precursor (m/z 341) inthe first quadrupole with (2) higher energy dissociation in thesecond quadrupole collision cell followed by (3) buildup ofthe desired low abundance m/z 221 product ion in the lineartrap, all three of which were necessary to obtain thesestructural details for 1–3 linked disaccharides.
Table 1. Spectral Similarity Scores for 1–3 Linked Disaccharides Versus Monosaccharide-GA Standards
Disaccharides Synthesized Standards
α-D-Glcp-GA β-D-Glcp-GA α-D-Galp-GA α-D-Manp-GA
α-D-Glcp-(1–3)-Glc 0.9977 0.8845 0.8823 0.7968β-D-Glcp-(1–3)-Glc 0.8572 0.9840 0.7608 0.8568α-D-Glcp-(1–3)-Fru 0.9930 0.8899 0.9027 0.8045α-D-Galp-(1–3)-Gal 0.9178 0.7530 0.9838 0.6942α-D-Manp-(1–3)-Man 0.8461 0.8702 0.7527 0.9891
356 C. Konda et al.: Structure Determination for Disaccharides
144
AcknowledgmentB.B. acknowledges support from the National ScienceFoundation CHE-0137986.
References1. Taylor, M.E., Drickamer, K.: Introduction to Glycobiology, 2nd edn.
Oxford University Press, Oxford (2006)2. Varki, A., Cummings, R.D., Esko, J.D., Freeze, H.H., Stanley, P.,
Bertozzi, C.R., Hart, G.W., Etzler, M.E.: Essentials of Glycobiology.Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2009)
3. Bush, C.A., Martin-Pastor, M., Imberty, A.: Structure and conformationof complex carbohydrates of glycoproteins, glycolipids, and bacterialpolysaccharides. Annu. Rev. Biophys. Biomolec. Struct. 28, 269–294(1999)
4. Bendiak, B., Fang, T.T., Jones, D.N.: An effective strategy for structuralelucidation of oligosaccharides through NMR spectroscopy combinedwith peracetylation using doubly 13C-labeled acetyl groups. Can. J.Chem. 80, 1032–1050 (2002)
5. Armstrong, G.S., Mandelshtam, V.A., Shaka, A.J., Bendiak, B.: Rapidhigh-resolution four-dimensional NMR spectroscopy using the filterdiagonalization method and its advantages for detailed structuralelucidation of oligosaccharides. J. Magn. Reson. 173, 160–168 (2005)
6. Zaia, J.: Mass spectrometry of oligosaccharides. Mass Spectrom. Rev.23, 161–227 (2004)
7. Fenn, J., Mann, M., Meng, C., Wong, S., Whitehouse, C.: Electrosprayionization for mass spectrometry of large biomolecules. Science 246,64–71 (1989)
8. Reinhold, V.N., Reinhold, B.B., Costello, C.E.: Carbohydrate molecularweight profiling, sequence, linkage, and branching data: ES-MS andCID. Anal. Chem. 67, 1772–1784 (1995)
9. Karas, M., Bachmann, D., Bahr, U., Hillenkamp, F.: Matrix-assistedultraviolet laser desorption of non-volatile compounds. Int. J. MassSpectrom. 78, 53–68 (1987)
10. Tanaka, K., Waki, H., Ido, Y., Akita, S., Yoshida, Y., Yoshida, T.,Matsuo, T.: Protein and polymer analyses up to m/z 100,000 by laserionization time-of-flight mass spectrometry. Rapid Commun. MassSpectrom. 2, 151–153 (1988)
11. Harvey, D.J.: Quantitative aspects of the matrix-assisted laser desorp-tion mass spectrometry of complex oligosaccharides. Rapid Commun.Mass Spectrom. 7, 614–619 (1993)
12. Domon, B., Costello, C.E.: A systematic nomenclature for carbohydratefragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconj.J. 5, 397–409 (1988)
13. Domon, B., Costello, C.E.: Structure elucidation of glycosphingolipidsand gangliosides using high-performance tandem mass spectrometry.Biochemistry 27, 1534–1543 (1988)
14. Garozzo, D., Giuffrida, M., Impallomeni, G., Ballistreri, A., Montaudo,G.: Determination of linkage position and identification of the reducingend in linear oligosaccharides by negative ion fast atom bombardmentmass spectrometry. Anal. Chem. 62, 279–286 (1990)
15. Dallinga, J.W., Heerma, W.: Reaction mechanism and fragment ionstructure determination of deprotonated small oligosaccharides, studiedby negative ion fast atom bombardment (tandem) mass spectrometry.Biol. Mass Spectrom. 20, 215–231 (1991)
16. Carroll, J.A., Ngoka, L., Beggs, C.G., Lebrilla, C.B.: Liquid secondaryion mass spectrometry/Fourier transform mass spectrometry of oligo-saccharide anions. Anal. Chem. 65, 1582–1587 (1993)
17. Mulroney, B., Traeger, J.C., Stone, B.A.: Determination of both linkageposition and anomeric configuration in underivatized glucopyranosyldisaccharides by electrospray mass spectrometry. J. Mass Spectrom. 30,1277–1283 (1995)
18. Guan, B., Cole, R.B.: MALDI linear-field reflectron TOF post-sourcedecay analysis of underivatized oligosaccharides: Determination ofglycosidic linkages and anomeric configurations using anion attach-ment. J. Am. Soc. Mass Spectrom. 19, 1119–1131 (2008)
19. Jiang, Y., Cole, R.B.: Oligosaccharide analysis using anion attachmentin negative mode electrospray mass spectrometry. J. Am. Soc. MassSpectrom. 16, 60–70 (2005)
20. Firdoussi, A.E., Lafitte, M., Tortajada, J., Kone, O., Salpin, J.-Y.:Characterization of the glycosidic linkage of underivatized disaccharidesby interaction with Pb2+ ions. J. Mass Spectrom. 42, 999–1011 (2007)
21. Staempfli, A., Zhou, Z., Leary, J.A.: Gas-phase dissociation mecha-nisms of dilithiated disaccharides: Tandem mass spectrometry andsemiempirical calculations. J. Org. Chem. 57, 3590–3594 (1992)
22. Laine, R.A., Pamidimukkala, K.M., French, A.D., Hall, R.W., Abbas,S.A., Jain, R.K., Matta, K.L.: Linkage position in oligosaccharides byfast atom bombardment ionization, collision-activated dissociation,tandem mass spectrometry and molecular modeling. L-Fucosylp-(↦1→X)-D-N-acetyl-D-glucosaminylp-(β1→3)-D-galactosylp-(β1-O-methyl) where X=3, 4, or 6. J. Am. Chem. Soc. 110, 6931–6939 (1988)
23. Hofmeister, G.E., Zhou, Z., Leary, J.A.: Linkage position determinationin lithium-cationized disaccharides: tandem mass spectrometry andsemiempirical calculations. J. Am. Chem. Soc. 113, 5964–5970 (1991)
24. Asam, M.R., Glish, G.L.: Tandem mass spectrometry of alkalicationized polysaccharides in a quadrupole ion trap. J. Am. Soc. MassSpectrom. 8, 987–995 (1997)
25. Polfer, N.C., Valle, J.J., Moore, D.T., Oomens, J., Eyler, J.R., Bendiak,B.: Differentiation of isomers by wavelength-tunable infrared multiple-photon dissociation-mass spectrometry: Application to glucose-contain-ing disaccharides. Anal. Chem. 78, 670–679 (2005)
26. Simoes, J., Domingues, P., Reis, A., Nunes, F.M., Coimbra, M.A.,Domingues, M.R.: Identification of anomeric configuration of under-ivatized reducing glucopyranosyl-glucose disaccharides by tandemmass spectrometry and multivariate analysis. Anal. Chem. 79, 5896–5905 (2007)
27. Zhang, H., Brokman, S.M., Fang, N., Pohl, N.L., Yeung, E.S.: Linkageposition and residue identification of disaccharides by tandem massspectrometry and linear discriminant analysis. Rapid Commun. MassSpectrom. 22, 1579–1586 (2008)
28. Mendonca, S., Cole, R., Zhu, J., Cai, Y., French, A., Johnson, G.,Laine, R.: Incremented alkyl derivatives enhance collision inducedglycosidic bond cleavage in mass spectrometry of disaccharides. J. Am.Soc. Mass Spectrom. 14, 63–78 (2003)
29. Gaucher, S.P., Leary, J.A.: Stereochemical differentiation of mannose,glucose, galactose, and talose using zinc(II) diethylenetriamine and ESI-ion trap mass spectrometry. Anal. Chem. 70, 3009–3014 (1998)
30. Desaire, H., Leary, J.A.: Differentiation of diastereomeric N-acetylhex-osamine monosaccharides using ion trap tandem mass spectrometry.Anal. Chem. 71, 1997–2002 (1999)
31. Mueller, D.R., Domon, B.M., Blum, W., Raschdorf, F., Richter, W.J.:Direct stereochemical assignment of sugar subunits in naturallyoccurring glycosides by low energy collision induced dissociation.Application to papulacandin antibiotics. Biol. Mass Spectrom. 15, 441–446 (1988)
32. Domon, B., Muller, D.R., Richter, W.J.: Determination of interglyco-sidic linkages in disaccharides by high performance tandem massspectrometry. Int. J. Mass Spectrom. Ion Process. 100, 301–311 (1990)
33. Fang, T.T., Bendiak, B.: The stereochemical dependence of unim-olecular dissociation of monosaccharide-glycolaldehyde anions in thegas phase: A basis for assignment of the stereochemistry and anomericconfiguration of monosaccharides in oligosaccharides by mass spec-trometry via a key discriminatory product ion of disaccharidefragmentation, m/z 221. J. Am. Chem. Soc. 129, 9721–9736 (2007)
34. Fang, T.T., Zirrolli, J., Bendiak, B.: Differentiation of the anomericconfiguration and ring form of glucosyl-glycolaldehyde anions in thegas phase by mass spectrometry: isomeric discrimination between m/z221 anions derived from disaccharides and chemical synthesis of m/z221 standards. Carbohydr. Res. 342, 217–235 (2007)
35. Brown, D.J., Stefan, S.E., Berden, G., Steill, J.D., Oomens, J., Eyler, J.R., Bendiak, B.: Direct evidence for the ring opening of monosaccha-ride anions in the gas phase: photodissociation of aldohexoses andaldohexoses derived from disaccharides using variable-wavelengthinfrared irradiation in the carbonyl stretch region. Carbohydr. Res.346, 2469–2481 (2011)
36. Carroll, J.A., Ngoka, L., Beggs, C.G., Lebrilla, C.B.: Liquid secondaryion mass spectrometry/Fourier transform mass spectrometry of oligo-saccharide anions. Anal. Chem. 65, 1582–1587 (1993)
37. Bendiak, B., Fang, T.T.: Assignment of the stereochemistry andanomeric configuration of structurally informative product ions derivedfrom disaccharides: infrared photodissociation of glycosyl-glycolalde-hydes in the negative ion mode. Carbohydr. Res. 345, 2390–2400(2010)
38. Wells, J.M., McLuckey, S.A. Collision-Induced Dissociation (CID) ofPeptides and Proteins. In: Biol. Mass Spectrom. Burlingame, A.L. (ed.)Academic Press, Amsterdam, pp. 148–185 (2005)
C. Konda et al.: Structure Determination for Disaccharides 357
145
39. Stein, S., Scott, D.: Optimization and testing of mass spectral librarysearch algorithms for compound identification. J. Am. Soc. MassSpectrom. 5, 859–866 (1994)
40. Frewen, B.E., Merrihew, G.E., Wu, C.C., Noble, W.S., MacCoss, M.J.:Analysis of peptide MS/MS spectra from large-scale proteomics experi-ments using spectrum libraries. Anal. Chem. 78, 5678–5684 (2006)
41. Lam, H., Deutsch, E.W., Eddes, J.S., Eng, J.K., King, N., Stein, S.E.,Aebersold, R.: Development and validation of a spectral librarysearching method for peptide identification from MS/MS. Proteomics7, 655–667 (2007)
42. Lam, H., Deutsch, E.W., Eddes, J.S., Eng, J.K., Stein, S.E., Aebersold,R.: Building consensus spectral libraries for peptide identification inproteomics. Nat. Methods 5, 873–875 (2008)
43. Kameyama, A., Kikuchi, N., Nakaya, S., Ito, H., Sato, T.,Shikanai, T., Takahashi, Y., Takahashi, K., Narimatsu, H.: Astrategy for identification of oligosaccharide structures usingobservational multistage mass spectral library. Anal. Chem. 77,4719–4725 (2005)
44. Zhang, Z.: Prediction of low-energy collision-induced dissociationspectra of peptides. Anal. Chem. 76, 3908–3922 (2004)
358 C. Konda et al.: Structure Determination for Disaccharides
146