IonSpec

FT ICR Isotopic Resolution of Proteins: Myoglobin

12 T qFT ICR MS (UWO)

MS/MS with FT ICR

1. SORI: Sustained off-resonance irradiation Excitation of selected ions slightly off- resonance. This results in ions being alternatively accelerated and decelerated limiting the cyclotron radius. Collision gas is introduced briefly to produce collision activated dissociation (CAD). The fragment ions are then produced close to the center and can be detected repetitively to give better sensitivity and resolution.

2. IRMPD: Infrared multi-photon dissociation

External IR laser provides energy to activate bonds and cause dissociation. Does not require introduction of gas pulses and therefore shortens the duty cycle

Fragmentation of peptides gives mostly b an y ions (as in CAD in quad)

Fragmentation of peptides gives mostly b an y ions (as in CAD in quad)

3. ECD: Electron Capture Dissociation

Heated filament inside the cell emits electrons that are capture by the ions inside the cell. The precursor ion must be doubly or morecharged to yield singly charged radical ion. Dissociation or fragmentation will occur a the amide bond between the NH and CH (N-C)giving primarily c and z ions. It is therefore complementary to SORIIRMPD and classical CAD in quad collision cell. Another advantage is for the analysis of PTMs since the amide bond is cleaved first not thephospho bond or glycosidic linkage.

H2N C C N

O

R1

HHC C N

O

R2

HHC C N

O

R3

HHC C N

O

R4

HHC C OH

O

R5

H

a1 b1 a2 a3 a4 a5 b2

b4 b3 c2 c3 c4c1

x1 y1 z1x4 x3 x2y4 y3 y2z3z4 z2

H2N C C N

R1

OH HHC C N

O

R2

HH

. H2N C C N

R1

OH HH HHC C N

O

R2

.

z

+

c

H2N C C N

R1

OHHH

C C N

O

R2

HH+

+ e-

81.972+

897.512+

998.552+

712.434+

948.032+

1104.612+

868.512+

1657.78493.31 1832.431187.66 1527.081054.58

2+1272.63

594.37 642.46541.673+

1927.20

0

20

40

60

80

100

400 600 800 1000 1200 1400 1600 1800 2000

712.184+

906.473+

1216.972+

949.113+

969.15

1415.282+

1721.44

1159.992+

1104.022+

656.23

528.211075.06

2+1127.03

863.833+

811.662+

1033.99 1656.25

1336.88 1830.70755.172+

838.641925.301358.84

2+

989.072+

414.15 612.24

1525.88

m/z

Electron capture dissociation (ECD) → c, z ions

Infrared multiphoton dissociation (IRMPD) → b, y ions

Complementary Sequencing from Two Different MS/MS TechniquesJLC

R. Zubarev, N. L. Kelleher, and F. W. McLafferty JACS 1998 120 (13) 3265

“Top-Down” Sequencing

Inject intact protein in the FT ICR and perform one or more (or combine) fragmentation method (ie ECD and ECD + IRMPD).

Mol. Cell. Proteomics 2003 2: 1253-1260

Advantages of Top-Down Sequencing

Can get the identity of the proteins from full sequence including thepost-translational modifications which are often missed by bottom-up approaches since coverage tends to be poor 5-30%.

Hybrid FTICR instruments with quadrupole for ion selection and CAD before entering the ICR cell: qFTMS (Bruker, IonSpec) and linear ion trap (LTQ FT, Thermo Finnigan); much more sensitive.

However:Size limitation ~45kDaLimit of detection: larger proteins are more difficult to ionize

Orbitrap

Latest on the market with a new type of mass analyzer: Hybrid instrument combining with a linear trap with the orbitrap

FT MS with no magnet!

FT MSImage current detectorHigh resolutionAccurate mass

2 Detectors in one instrument

FT MS with no magnet!

Orbitrap

Detection and Measurement of Ions in the Orbitrap

The orbitrap mass analyzer employs the trapping of pulsed ion beams in an electrostatic quadro-logarithmic field. This field is created between an axial central electrode and a coaxial outer electrode. Stable ion trajectories combine rotation around the central electrode with harmonic oscillations along it. The frequencies of axial oscillations and hence mass-to-charge ratios of ions are obtained using fast Fourier transform of the image current detected on the two split halves of the outer electrode.

Hardman M, Makarov AA. Anal. Chem. 2003 Apr 1;75(7):1699-705.

Orbitrap: summary

Excellent mass accuracy especially with internal standard < 1.0 ppm*: mass accuracy is critical for precursor ion

Can perform MS/MS in linear trap and fragments analyzed i n either theLinear trap (faster but lower resolution) or in the Orbitrap.

Important new tool in “bottom-up” proteomics

Disadvantages:

Poor for intact proteins Cannot do MS/MS by IRMPD, ECD, etc.

* M. Mann et al. Mol. Cell. Proteomics 2005, 2010

Quantitation by MS

-Use isotopes enrichment:- ICAT, cleavable ICAT, iTRAQ

- Labeling with N15, C13

- Labeling with O18 (With protease digestion)

- Labeled amino acid added to growth media (SILAC) Leu (d3), Ser, Tyr, Lys (d4), Arg (13C, 15N)

- Internal calibrant with isotoptically labeled peptide (AQUA)

-Others: 1. add known amount of protein to mixture and digested and/ or normalized against proteins that do not change in intensity

LC MS is not strictly quantitative: different peptides ionize differently in both MALDI and ESI: Need internal calibrants most often with isotope enriched similar chemical species

2. Intensity of signal (normalized)

Quantitative Proteomic Profiling

Stable Isotope labeling2D GE Intensity-based quantitation

In vivo labeling In vitro labeling

N-terminalpeptide labeling

C-terminalpeptide lableling

Amino Acid-Based labeling

N14/N15 media SILAC

Cys:ICATiTRAQ

Esterification(CD3OH)

16O/18O incorporationvia proteolysis

To quantitate expression of proteins from cells exposed to differentconditions by ESMS (ion trap) and avoid problematic 2D gels:

Add an affinity label- biotin (avidin tight binding) linked by a short spacer which contain either 0 or 8 deuteriums and alkylating portion to react with SH of cysteines.

Affinity chromatography: retains only cysteines labeled peptides

Measure relative intensities of peaks separated by 8 Da. Peptides only differ by deuterium and separate and ionize the same.

Improvement in separation affinity column (30 fractions) each ion exchange (30 fractions) and RP HPLC (30 fractions) 2700 samples to analyze by ESMS !!

ICAT: Isotope Encoded Affinity Tags

Original ICAT Reagent

HN NH

O

X

SNH

O

OX

XX

OO N

X

XX

X

O

I

H

Gygi et al, Nature Biotech (1999) 17 994Aebersold et al, Curr. Opin. Biotech (2003) 14, 110

Light ICAT d0 , X = H

Heavy ICAT d8, X = D

HS-Cys-peptide

Biotin Affinity tag Linker with labels Reactive group

ICAT Procedure

1. Denature (urea +SDS) and reduce proteins (TCEP) (Tris- caboxyethylphophine) HCl2. Incubate with ICAT reagent

3. Combine control and test samples

4. Digest with trypsin

5. Chromatography: A. Affinity Biotin/Avidin: rejects non labelled peptides, simplifying mixture B. Reverse Phase HPLC

7. MS to measure relative intensity of peaks differing by 8 Da and MS/MS to ID peptides

HS SHSH

SHSH HS

SH

HS

HS SHSH

SHSH HS

SH

HS

SSSSSSS

S

SSS

S S S

SSSSSSS

S

SSS

S S S

Denature and reduce

Labeling with light ICAT Labeling with heavy ICAT

Mix and Trypsin digestion

S

SS

S

S

S

S

SS

S

S

S

Affinity capture biotin/avidin

LC MS MS/MS RatioLight /heavy ID

Non retained

Peptides

ICAT (cont..)

m/z m/z

LC MS

S

S

8 Da

MS/MS

Database Search

(MASCOT)

Identification of protein

Ratio ~ 60:40

L

H

560 568

(281.5)

Select doubly charged ion

Limitations:

1) does not work with proteins with no cysteine (in yeast 20%, more in other organisms)

2) only identify small peptide portion of these proteins ie miss post-translational modifications

3) limited MS/MS sequencing because interference of the ICAT label

4) Long deuterated PEG chain changes HPLC retention time. The intensity in the MS is not accurate to quantitate both species (ie arrive at different times at the MS).

ICAT Isotope encoded affinity tags

Isotope tag X = H or D (m = 7)

Zhou et al., Nature Biotech. 2002, 19, 512

Solid Phase ICAT

Glass beadsPhotocleavable linker

Thiol reactive

CXX3C CX3

H

H

H

h cleavageHS-Cys-peptide

N

O

O

CH3 N

O

N

O

I

OCH3

NO2

Better, but still problems: reagent is very light sensitive (and expensive)

Quantitation: Labeling with O18

Procedure:

On one test sample extract proteins, reduce, alkylate cysteines and treated with trypsin in the presence of H2O18 : get incorporation of O18 at the C terminal carboxylic acid (from attack of H2O18 on the acyl-enzyme intermediate

Mix this digest with tryptic digests the control samplesPerformed in the presence of H2O. Perform MS and measure relative intensity of peaks differing by 2 Da.

One can get incorporation of 2 O18 simplifying the analysis by promoting trypsin catalyzed second exchange. Works better with Arg than for Lys

Quantitation: Labeling with O18

Mass difference of 2 (or 4): need high resolution MS !

Get 1 (or 2 O18)

m/z

Inte

nsity

COH

O

Arg (Lys)

18

18

COH

O

Arg (Lys)

18

COH

O

Arg (Lys)

+2 +4

MS of tryptic peptides digest labeled with O18 vs control

100%

Ratio labeled vs unlabeled ~2:1

With Arg can get exclusively + 4 DaWith Lys get a mixture of + 2 and + 4 Da

Labeled amino acid added to growth media Leu(d10), Ser (d3), Tyr (d2), Lys (d4), Arg (N14, C13) With auxotrophs strains or with media depleted in these amino acids

Quantitation: Labeled Amino Acids

Limitations: Some ScramblingNot generally applicable to all types of experimtns (eg Human tissues)

J. Proteome Research 2002,1 , 345-350Rapid Comm. Mass Spec. 2002,16 2115-2123

SILAC (stable isotope labeling with amino acidsIn cell culture)

Leu-d3

SILAC Procedure

Ong, S.-E. (2002) Mol. Cell. Proteomics 1: 376-386

Incorporation of Leu-d3 in proteins at various time points

Ong, S.-E. (2002) Mol. Cell. Proteomics 1: 376-386

The use of dialyzed serum avoids nonspecific incorporation of non-labeled leucine derived from serum in the Leu-d3 samples

Ong, S.-E. (2002) Mol. Cell. Proteomics 1: 376-386

Quantitation of nine proteins during C2C12 cell differentiation by SILAC

Ong, S.-E. (2002) Mol. Cell. Proteomics 1: 376-386

Observed ratios are similar to expected ratios in mixing experiments

Labeling with 15N or 13C

Gives complex mixturedifficult to interpret

Each peptide is enrichedwith 15N and/or 13C

Most useful in MS only,with FT ICR MS and“accurate mass tag”

Quantitation in the MS/MS: the iTRAQ approach

iTRAQ: Isotope Tags Reagents for Accurate Quantitation

Quantitation for drugs and metabolites are usually done in the MS/MS mode: to minimize chemical noise and get better accuracy.

Design sets of chemicals tags to react with N-terminal amines (and Lysine)that would have the same retention times on LC AND the same mass (isobaric) in the MS experiments but for which fragments (reporter) would differ in the MS/MS.

NH -peptide

Reporter group114,115,116.117

Balance group31,30,29,28

MS/MS Fragmentation site

Isobaric tag mass = 142

N N

N

CH3

CH3

O

Y Y= leaving group

N N

N

CDH2

H2DC

O

Y

N N

N

CH3

H2DC

O

Y

N N

N

CD2H

H2DCO

Y

*

*

*

*

114 115

116117

31 30

2928

The Four iTRAQ Reagents

N N

N

O

HN

R

O

OH*

*

N N

N

C

O

HN

R

O

OH

*

*+

CID

PeptideFragments

Quantifying Fragment

m/z 114 32 Da Peptide Identifciation

iTRAQ: Procedure

Up to 4 test samples

ITRAQ: Advantages and Disadvantages

-Can perform up to four quantitation experiment at once. Allow for time course experiments e.g. 0 min, 10 min, 30 min, 60 min each with a different label

-The reporter group does not interfere with the peptide fragmentation so that quantitation and peptide ID can be done in the same experiment.

Advantages

Disadvantages

-React with other amine groups ie lysine side chain and amines in buffer. The amine containing buffers have to be replaced before labeling.

- Very expensive reagents!

Post-translational modifications (PTMs)

Modifications are very important sometime determinant for function (over 200 known modifications)

Most important:

Phosphorylation +80 Enzyme Activity, signalingpTyr, +++pSer, pThr +/++Glycosylation Cell/cell recognition N-linked >800 ++ O-Linked >203 ++Acetylation +42 +++ Protein stabilityMethylation +14 +++ Gene expressionAcylation Cellular Localization Farnesyl +204 +++ Myristoyl +210 +++

Sulfation +80 Protein/protein interactionsDisulfide -2 ++ Protein stabilityDeamidation +1 +++ Protein/protein interactions, agingUbiquitination >1000 +++ Protein degradationNitration +45 ++ Oxidative damageTruncation -x +++

Detection of Phosphopeptides at Low Levels

Phosphorylation: > 100,000 possible sites in human; ~2000 known ~1/3 of human proteins are phosphorylated

Notoriously difficult to analyze, especially at low levels. sub-stoichiometrypoor ionization and ion suppressionother factors? absorption on LC columns

Multiply phosphorylated are not observed

What factors are responsible for the absorption of phosphopeptides, especially mutliply-phosphorylated peptides on LC?

Complexation of phosphate groups with free Si-OH on C18 RP material?

Smith R. D. et al (2004) J. Mass Spectrom. 39:208-215

Several approaches for enrichment have been described:

• IMAC with Fe+3 (or Ga+3, Al+3) and variations followed by LC MS •Beta-elimination/addition of thiols

• Graphite columns to retain the “very hydrophilic phosphopeptides”

• Addition of H3PO4 to the sample to elute multiply-phosphorylated peptides bound to silica on the RP column*

•TiO2 columns for enrichment

* Smith R. D. et al (2004) J. Mass Spectrom. 39:208-215

Detection of Phosphopeptides at Low Levels

Post-translational modifications (PTMs): Phosphorylation

Phosphorylation: 100,000 possible sites in human; ~2000 known ~1/3 of human proteins are phosphorylated. One of the main challenge in proteomics

D. Kalume et al Curr. Opinion in Chem. Biol. 2003, 7:64-69

pTyr: (~1%) Chemically stable: observed +80 enrichment with phospho Tyr specific antibodies or with IMAC (immobilized metal affinity chromatography)

pThr: (~10%) -less stable but can be observed in MS

pSer: (~90%) Unstable: usually undergoes by base or in MS -elimination to dehydro-Ala (-98) which can react with nucleophile (thiols, amines) enrichment by IMAC

Immobilized Metal Affinity Chromatography: IMAC

Based on affinity of phosphates to certain metals Fe+3,Ga+3

Phosphopeptides are preferentially retained but so are strongly acidic peptides (Asp, Glu)

Solution: esterify with MeOH/ HCl prior to column

Phosphorylation can be verified by alkaline phosphatase Lost of 80 Da by MS

Note: Phosphopeptides tend to inonize less efficiently than their non-phosphorylated counterpart

N

H2C

H2C

C

C

O

O

O

O

M CH

C

CH2

O

OP

OH

O

O

NH

CH

C

CH3

OH

O

HN

CH

C

H2C

HOO

OP

OH

O

O

IMAC

M = Fe , Ga

Phosphopeptides: after IMAC

Modification: esterification of carboxylates improves sensitivity

Nat. Biotech. 2002, 20, 301

Location of Phosphorylated Residue(s) by MS/MS

Select precursor ion (+80) for MS/MS

PhosphoTyrosine: The phosphate is stable : look for difference of 243 in between the y (or b) ions (Tyr 163 + Phosphate 80)

PhosphoSerine: The phosphate is very labile and is lost in the collision of the MS/MS experiment resulting in a dehydroalanine residue: the peptide bond is then fragmented. The location can be determined by the difference of 69

between 2 y adjacent (or b) ions

PhosphoThreonine: The phosphate is partially lost followed by peptide bond fragmentation. The location of 83 between adjacent y (or b) ions. This is most often accompanied by series of ions containing intact phosphothreonine. Therefore the location can be determined by mass difference of 181 (Thr 101 + 80)

NH

OPO3H2

O

NHH

R

:Base

NH

O

NH

R

+ H3PO4

-Elimination

-98

NH

OH

O

NHH

R

O

Ba(OH)2

-69

MS/MS

-87

Dehydroalanine

Serine

Phosphoserine and -elimination

Phosphate group of serine is very labile and can be -eliminated by base treatment OR in the MS experiment

NH

O

NH

R

+ H3PO4

69

yx y*x+1

NH

OH

O

NHH

R

O

87

yx yx+1

MS/MS of Phosphoserine vs Serine

m/zm/z

dehydroalanineserine

NH

OPO3H2

O

NHH

R

MS/MS of PhosphoThreonine vs Threonine

83

yx y*x+1

101

yx yx+1

m/z

yx+1

181

Mixture of both species

-181

NH

O

NH

R

OH

NH

O

NH

R+ H3PO4

- 83 -101

MS Detection of Phosphopeptides

Several ways by ESI:

- Neutral loss scan: (–98) with triple quad or linear trap. Once theprecursor ion is identified can perform MS/MS and identify peptide and site of phosphorylation.

- FT ICR and ECD:

-Precursor ion scan: monitor loss of phosphate ion in negative ion mode

Cleavages at amide bonds leaving phospophates attached to amino acids.

Easier to determine sites of phosphorylation

Phospho-Specific Hydrolysis of Phosphopeptides

Converts phophopeptides to aminoethylcysteines by -elimination (to dehydro-Ala) followed by addition of aminoethylthiol: get both R and S isomer

Treat derivatized proteins with Lys-C or trypsin:get cleavage at Lys sites and at phosphorylation sites(now present at aminoethylcysteine)

Analysis by MS and MS/MS clearly identifies phosphorylation sites. Only the S isomer is cleaved by the enzyme

Nature Biotech. 2003 21, 1047 - 1054

NH

O

NH

R

HSCH2CH2NH2

H

NH

O

NH

RH

S

NH2

NH

O

NH

RH

NH2

D,L-Lysine

NH

O

S

OHH

NH2

NH

O

NH

R

S

NH2

D

H

L (only)

Trypsin

Phospho-Specific Hydrolysis of Phosphopeptides

Shokat, Nature Biotech 2003, 21 1047

Nature Biotech. 2003 21, 1047-1054

Phospho-Specific Hydrolysis of Phosphopeptides

Trypsin H216O Trypsin H2

18O

O18

O18

O18

IMAC

Phosphatase

O18

O18

4 Da

PNAS 2003 100(3) 880.

Quantitation of Phosphoproteins

Control Experiment

~ 60~ 40

Time Course Study of Phosphorylation with SILAC

Nature Biotech 2004 22(9) 1139

Nature Biotech 2004 22(9) 1139

Time Course Study of Phosphorylation

PTMs: Glycoproteins

Much more complex:

• O-linked (Ser/Thr), N-Linked (Asn), or both

• Microheterogeneity: same protein may have different glycoforms

PTMs : Glycoproteins

Can use speficic enzymes to determine nature of sugars and linkages (a,b) :

-mannosidase-glycosidase,etc..

O-linked: -can undergo b-elimination: complicates pSer/pThr analysis) -no specific O-glycanase to cleave from Se/Thr

N-linked: -Can remove entire sugar with PNGase F (Asn to Asp)

-EndoF cleaves between the first two sugars

HO

O

OOH

OH

HO

O

OOH

OH

HO

O

OOH

OH

HO

O

OOH

OH

HO

Non reducing end

A1 B1 B3B2B4

Y1 Y4Y3Y2

C1 C2 C3 C4

Z1 Z2 Z3 Z4

Reducing end

Glycoproteins: most common fragmentation

Use analogous nomenclature to peptides with capital lettersY, B, C, Z, etc

In certain conditions, can get “cross-ring” fragmentation (A1)

MS/MS of Glycopeptide from RNAse B

994.69

a)163.0 b)457.15 c)660.24 d)1009.36

e)990.43 f)997.94

g)1222.21 h)1154.52 i)1038.15

j)994.43

1031.21961.68

990.69

1173.87163.06 1282.25

1222.54a

c

j

h

457.15b 1038.47

i

e

998.19f

g

994.69

a)163.0 b)457.15 c)660.24 d)1009.36

e)990.43 f)997.94

g)1222.21 h)1154.52 i)1038.15

j)994.43

a)163.0 b)457.15 c)660.24 d)1009.36

e)990.43 f)997.94

g)1222.21 h)1154.52 i)1038.15

j)994.43

1031.21961.68

990.69

1173.87163.06 1282.25

1222.54a

c

j

h

457.15b 1038.47

i

e

998.19f

g

MS/MS fragmentation of Glycopeptides

(Peptide) 2+

(Peptide+HexNAc) 2+

Pentose (P) Hexose (H ) HexNAc (N) Fucose (F)

 

N2H3FX  

N2H2NFX  

N2H3NFX  

N2H3N2FX  

N2H3NHF2X  

N2H3N2HF2X  

N2H3N2H2F3X  

N2H3N3H3F2X  

N2H3N3H3F4X  

Pentose (P) Mannose(M) Hexose (H) HexNAc (N) Fucose (F)

Pentose (P) 132.042Deoxyhexose (F) 146.057

Hex (H) 162.052HexNAc (N) 203.079

1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 1 1 0 0 1 2 0 0 1 3 0 0 1 4 0 0 1 5 0 0m / z0

1 0 0

%

x 1 02 7 4 . 1 0

2 0 4 . 0 9

2 9 2 . 1 2

4 9 5 . 2 04 5 4 . 1 8

3 6 6 . 1 6

2 9 3 . 1 2 3 6 7 . 1 6

6 5 7 . 2 6

4 9 6 . 2 0

5 8 3 . 2 31 0 9 8 . 5 51 0 1 8 . 0 56 5 8 . 2 7

9 4 8 . 3 89 1 6 . 5 18 2 9 . 7 4 1 2 4 4 . 6 7 1 3 2 5 . 1 6

b 44 2 9 . 2

N

S

N H

H S N S

N H S

N H S 2

N

HS

S

T * ( p e p t i d e )2 9 2 . 1 + 4 5 4 . 2 +

9 4 8 . 3 +

1 1 4 2 . 5 + +

1 3 9 0 . 2 + +1 4 7 1 . 1 + +

7 2 0 7 4 0 7 6 0 7 8 0 8 0 0 8 2 0 8 4 0 8 6 0 8 8 0 9 0 0 9 2 0 9 4 0 9 6 0 9 8 0 1 0 0 0m / z0

1 0 0

%

9 4 8 . 3 8

9 1 6 . 5 18 7 2 . 4 78 2 9 . 7 4

7 7 0 . 4 47 1 0 . 4 4

7 1 2 . 9 5 7 3 3 . 0 17 1 4 . 4 3 7 4 6 . 2 7

8 1 4 . 4 37 7 1 . 4 6 7 8 7 . 0 6

7 9 4 . 4 6

8 3 0 . 0 8 8 7 1 . 9 6

8 5 8 . 9 48 3 0 . 7 4

8 5 1 . 4 9

8 8 4 . 1 2 9 1 5 . 9 78 8 4 . 4 6

8 9 5 . 5 19 1 4 . 5 3

9 4 0 . 0 0

9 6 0 . 5 1

9 9 7 . 5 2

9 8 5 . 5 39 6 0 . 9 8

9 6 1 . 5 39 9 8 . 0 5

b 7

2 +P e p + N

2 +b 1 2 + N 2/ ( y 1 2 + N 2 )

3 +P e p + N 2 H S 2

2 +y 1 2 + N S

2 +P e p + N 2

3 +P e p + N 2 H 2 S 2

2 +P e p + N S

1 +N H S 2

2 +y 1 2 + N 2 S

2 +P e p + N 2 H

2 +P e p + N H S

1 0 2 5 1 0 5 0 1 0 7 5 1 1 0 0 1 1 2 5 1 1 5 0 1 1 7 5 1 2 0 0 1 2 2 5 1 2 5 0 1 2 7 5 1 3 0 0 1 3 2 5 1 3 5 0 1 3 7 5 1 4 0 0 1 4 2 5 1 4 5 0 1 4 7 5m / z0

1 0 0

%

1 0 9 8 . 5 51 0 7 9 . 5 51 0 1 8 . 0 5

1 0 1 8 . 5 31 0 7 8 . 4 5

1 0 7 8 . 2 0

1 0 7 7 . 2 2

1 0 4 2 . 0 51 0 7 5 . 2 4

1 0 7 2 . 5 3

1 0 9 9 . 5 91 2 4 4 . 6 7

1 2 4 4 . 1 4

1 1 6 3 . 6 11 1 0 0 . 0 7

1 1 6 3 . 1 0

1 1 2 2 . 6 6

1 1 8 6 . 6 11 1 8 7 . 5 8

1 1 8 8 . 0 8

1 2 3 0 . 6 0

1 3 2 5 . 1 6

1 2 6 8 . 1 21 2 6 8 . 6 11 2 6 9 . 2 1

1 3 9 0 . 1 91 3 2 6 . 6 01 3 8 9 . 6 3 1 4 1 3 . 7 0 1 4 7 1 . 0 9

2 +P e p + N 2 S

2 +y 1 2 + N 2 H S

2 +P e p + N 2 H S

2 +y 1 2 + N 2 H 2 S

2 +P e p + N 2 S 2

2 +P e p + N 2 H 2 S

2 +y 1 2 + N 2 H S 2

2 +P e p + N 2 H 2 S 2

2 +y 1 2 + N 2 H 2 S 2 +

y 1 2 + N 2 H 2 S 2

2 +P e p + N 2 H S 2

2 +P e p + N 2 H S 3

2 +P e p + N 2 H 2 S 3

A

C

B

1 +P e p

1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 1 1 0 0 1 2 0 0 1 3 0 0 1 4 0 0 1 5 0 0m / z0

1 0 0

%

x 1 02 7 4 . 1 0

2 0 4 . 0 9

2 9 2 . 1 2

4 9 5 . 2 04 5 4 . 1 8

3 6 6 . 1 6

2 9 3 . 1 2 3 6 7 . 1 6

6 5 7 . 2 6

4 9 6 . 2 0

5 8 3 . 2 31 0 9 8 . 5 51 0 1 8 . 0 56 5 8 . 2 7

9 4 8 . 3 89 1 6 . 5 18 2 9 . 7 4 1 2 4 4 . 6 7 1 3 2 5 . 1 6

b 44 2 9 . 2

N

S

N H

H S N S

N H S

N H S 2

N

HS

S

T * ( p e p t i d e )2 9 2 . 1 + 4 5 4 . 2 +

9 4 8 . 3 +

1 1 4 2 . 5 + +

1 3 9 0 . 2 + +1 4 7 1 . 1 + +

7 2 0 7 4 0 7 6 0 7 8 0 8 0 0 8 2 0 8 4 0 8 6 0 8 8 0 9 0 0 9 2 0 9 4 0 9 6 0 9 8 0 1 0 0 0m / z0

1 0 0

%

9 4 8 . 3 8

9 1 6 . 5 18 7 2 . 4 78 2 9 . 7 4

7 7 0 . 4 47 1 0 . 4 4

7 1 2 . 9 5 7 3 3 . 0 17 1 4 . 4 3 7 4 6 . 2 7

8 1 4 . 4 37 7 1 . 4 6 7 8 7 . 0 6

7 9 4 . 4 6

8 3 0 . 0 8 8 7 1 . 9 6

8 5 8 . 9 48 3 0 . 7 4

8 5 1 . 4 9

8 8 4 . 1 2 9 1 5 . 9 78 8 4 . 4 6

8 9 5 . 5 19 1 4 . 5 3

9 4 0 . 0 0

9 6 0 . 5 1

9 9 7 . 5 2

9 8 5 . 5 39 6 0 . 9 8

9 6 1 . 5 39 9 8 . 0 5

b 7

2 +P e p + N

2 +b 1 2 + N 2/ ( y 1 2 + N 2 )

3 +P e p + N 2 H S 2

2 +y 1 2 + N S

2 +P e p + N 2

3 +P e p + N 2 H 2 S 2

2 +P e p + N S

1 +N H S 2

2 +y 1 2 + N 2 S

2 +P e p + N 2 H

2 +P e p + N H S

1 0 2 5 1 0 5 0 1 0 7 5 1 1 0 0 1 1 2 5 1 1 5 0 1 1 7 5 1 2 0 0 1 2 2 5 1 2 5 0 1 2 7 5 1 3 0 0 1 3 2 5 1 3 5 0 1 3 7 5 1 4 0 0 1 4 2 5 1 4 5 0 1 4 7 5m / z0

1 0 0

%

1 0 9 8 . 5 51 0 7 9 . 5 51 0 1 8 . 0 5

1 0 1 8 . 5 31 0 7 8 . 4 5

1 0 7 8 . 2 0

1 0 7 7 . 2 2

1 0 4 2 . 0 51 0 7 5 . 2 4

1 0 7 2 . 5 3

1 0 9 9 . 5 91 2 4 4 . 6 7

1 2 4 4 . 1 4

1 1 6 3 . 6 11 1 0 0 . 0 7

1 1 6 3 . 1 0

1 1 2 2 . 6 6

1 1 8 6 . 6 11 1 8 7 . 5 8

1 1 8 8 . 0 8

1 2 3 0 . 6 0

1 3 2 5 . 1 6

1 2 6 8 . 1 21 2 6 8 . 6 11 2 6 9 . 2 1

1 3 9 0 . 1 91 3 2 6 . 6 01 3 8 9 . 6 3 1 4 1 3 . 7 0 1 4 7 1 . 0 9

2 +P e p + N 2 S

2 +y 1 2 + N 2 H S

2 +P e p + N 2 H S

2 +y 1 2 + N 2 H 2 S

2 +P e p + N 2 S 2

2 +P e p + N 2 H 2 S

2 +y 1 2 + N 2 H S 2

2 +P e p + N 2 H 2 S 2

2 +y 1 2 + N 2 H 2 S 2 +

y 1 2 + N 2 H 2 S 2

2 +P e p + N 2 H S 2

2 +P e p + N 2 H S 3

2 +P e p + N 2 H 2 S 3

A

C

B

1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 1 1 0 0 1 2 0 0 1 3 0 0 1 4 0 0 1 5 0 0m / z0

1 0 0

%

x 1 02 7 4 . 1 0

2 0 4 . 0 9

2 9 2 . 1 2

4 9 5 . 2 04 5 4 . 1 8

3 6 6 . 1 6

2 9 3 . 1 2 3 6 7 . 1 6

6 5 7 . 2 6

4 9 6 . 2 0

5 8 3 . 2 31 0 9 8 . 5 51 0 1 8 . 0 56 5 8 . 2 7

9 4 8 . 3 89 1 6 . 5 18 2 9 . 7 4 1 2 4 4 . 6 7 1 3 2 5 . 1 6

b 44 2 9 . 2

N

S

N H

H S N S

N H S

N H S 2

N

HS

S

T * ( p e p t i d e )2 9 2 . 1 + 4 5 4 . 2 +

9 4 8 . 3 +

1 1 4 2 . 5 + +

1 3 9 0 . 2 + +1 4 7 1 . 1 + +

1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 1 1 0 0 1 2 0 0 1 3 0 0 1 4 0 0 1 5 0 0m / z0

1 0 0

%

x 1 02 7 4 . 1 0

2 0 4 . 0 9

2 9 2 . 1 2

4 9 5 . 2 04 5 4 . 1 8

3 6 6 . 1 6

2 9 3 . 1 2 3 6 7 . 1 6

6 5 7 . 2 6

4 9 6 . 2 0

5 8 3 . 2 31 0 9 8 . 5 51 0 1 8 . 0 56 5 8 . 2 7

9 4 8 . 3 89 1 6 . 5 18 2 9 . 7 4 1 2 4 4 . 6 7 1 3 2 5 . 1 6

b 44 2 9 . 2

N

S

N H

H S N S

N H S

N H S 2

N

HS

S

T * ( p e p t i d e )2 9 2 . 1 + 4 5 4 . 2 +

9 4 8 . 3 +

1 1 4 2 . 5 + +

1 3 9 0 . 2 + +1 4 7 1 . 1 + +

N

HS

S

T * ( p e p t i d e )N

HS

S

T * ( p e p t i d e )2 9 2 . 1 + 4 5 4 . 2 +

9 4 8 . 3 +

1 1 4 2 . 5 + +

1 3 9 0 . 2 + +1 4 7 1 . 1 + +

7 2 0 7 4 0 7 6 0 7 8 0 8 0 0 8 2 0 8 4 0 8 6 0 8 8 0 9 0 0 9 2 0 9 4 0 9 6 0 9 8 0 1 0 0 0m / z0

1 0 0

%

9 4 8 . 3 8

9 1 6 . 5 18 7 2 . 4 78 2 9 . 7 4

7 7 0 . 4 47 1 0 . 4 4

7 1 2 . 9 5 7 3 3 . 0 17 1 4 . 4 3 7 4 6 . 2 7

8 1 4 . 4 37 7 1 . 4 6 7 8 7 . 0 6

7 9 4 . 4 6

8 3 0 . 0 8 8 7 1 . 9 6

8 5 8 . 9 48 3 0 . 7 4

8 5 1 . 4 9

8 8 4 . 1 2 9 1 5 . 9 78 8 4 . 4 6

8 9 5 . 5 19 1 4 . 5 3

9 4 0 . 0 0

9 6 0 . 5 1

9 9 7 . 5 2

9 8 5 . 5 39 6 0 . 9 8

9 6 1 . 5 39 9 8 . 0 5

b 7

2 +P e p + N

2 +b 1 2 + N 2/ ( y 1 2 + N 2 )

3 +P e p + N 2 H S 2

2 +y 1 2 + N S

2 +P e p + N 2

3 +P e p + N 2 H 2 S 2

2 +P e p + N S

1 +N H S 2

2 +y 1 2 + N 2 S

2 +P e p + N 2 H

2 +P e p + N H S

7 2 0 7 4 0 7 6 0 7 8 0 8 0 0 8 2 0 8 4 0 8 6 0 8 8 0 9 0 0 9 2 0 9 4 0 9 6 0 9 8 0 1 0 0 0m / z0

1 0 0

%

9 4 8 . 3 8

9 1 6 . 5 18 7 2 . 4 78 2 9 . 7 4

7 7 0 . 4 47 1 0 . 4 4

7 1 2 . 9 5 7 3 3 . 0 17 1 4 . 4 3 7 4 6 . 2 7

8 1 4 . 4 37 7 1 . 4 6 7 8 7 . 0 6

7 9 4 . 4 6

8 3 0 . 0 8 8 7 1 . 9 6

8 5 8 . 9 48 3 0 . 7 4

8 5 1 . 4 9

8 8 4 . 1 2 9 1 5 . 9 78 8 4 . 4 6

8 9 5 . 5 19 1 4 . 5 3

9 4 0 . 0 0

9 6 0 . 5 1

9 9 7 . 5 2

9 8 5 . 5 39 6 0 . 9 8

9 6 1 . 5 39 9 8 . 0 5

b 7

2 +P e p + N

2 +b 1 2 + N 2/ ( y 1 2 + N 2 )

3 +P e p + N 2 H S 2

2 +y 1 2 + N S

2 +P e p + N 2

3 +P e p + N 2 H 2 S 2

2 +P e p + N S

1 +N H S 2

2 +y 1 2 + N 2 S

2 +P e p + N 2 H

2 +P e p + N H S

1 0 2 5 1 0 5 0 1 0 7 5 1 1 0 0 1 1 2 5 1 1 5 0 1 1 7 5 1 2 0 0 1 2 2 5 1 2 5 0 1 2 7 5 1 3 0 0 1 3 2 5 1 3 5 0 1 3 7 5 1 4 0 0 1 4 2 5 1 4 5 0 1 4 7 5m / z0

1 0 0

%

1 0 9 8 . 5 51 0 7 9 . 5 51 0 1 8 . 0 5

1 0 1 8 . 5 31 0 7 8 . 4 5

1 0 7 8 . 2 0

1 0 7 7 . 2 2

1 0 4 2 . 0 51 0 7 5 . 2 4

1 0 7 2 . 5 3

1 0 9 9 . 5 91 2 4 4 . 6 7

1 2 4 4 . 1 4

1 1 6 3 . 6 11 1 0 0 . 0 7

1 1 6 3 . 1 0

1 1 2 2 . 6 6

1 1 8 6 . 6 11 1 8 7 . 5 8

1 1 8 8 . 0 8

1 2 3 0 . 6 0

1 3 2 5 . 1 6

1 2 6 8 . 1 21 2 6 8 . 6 11 2 6 9 . 2 1

1 3 9 0 . 1 91 3 2 6 . 6 01 3 8 9 . 6 3 1 4 1 3 . 7 0 1 4 7 1 . 0 9

2 +P e p + N 2 S

2 +y 1 2 + N 2 H S

2 +P e p + N 2 H S

2 +y 1 2 + N 2 H 2 S

2 +P e p + N 2 S 2

2 +P e p + N 2 H 2 S

2 +y 1 2 + N 2 H S 2

2 +P e p + N 2 H 2 S 2

2 +y 1 2 + N 2 H 2 S 2 +

y 1 2 + N 2 H 2 S 2

2 +P e p + N 2 H S 2

2 +P e p + N 2 H S 3

2 +P e p + N 2 H 2 S 3

1 0 2 5 1 0 5 0 1 0 7 5 1 1 0 0 1 1 2 5 1 1 5 0 1 1 7 5 1 2 0 0 1 2 2 5 1 2 5 0 1 2 7 5 1 3 0 0 1 3 2 5 1 3 5 0 1 3 7 5 1 4 0 0 1 4 2 5 1 4 5 0 1 4 7 5m / z0

1 0 0

%

1 0 9 8 . 5 51 0 7 9 . 5 51 0 1 8 . 0 5

1 0 1 8 . 5 31 0 7 8 . 4 5

1 0 7 8 . 2 0

1 0 7 7 . 2 2

1 0 4 2 . 0 51 0 7 5 . 2 4

1 0 7 2 . 5 3

1 0 9 9 . 5 91 2 4 4 . 6 7

1 2 4 4 . 1 4

1 1 6 3 . 6 11 1 0 0 . 0 7

1 1 6 3 . 1 0

1 1 2 2 . 6 6

1 1 8 6 . 6 11 1 8 7 . 5 8

1 1 8 8 . 0 8

1 2 3 0 . 6 0

1 3 2 5 . 1 6

1 2 6 8 . 1 21 2 6 8 . 6 11 2 6 9 . 2 1

1 3 9 0 . 1 91 3 2 6 . 6 01 3 8 9 . 6 3 1 4 1 3 . 7 0 1 4 7 1 . 0 9

2 +P e p + N 2 S

2 +y 1 2 + N 2 H S

2 +P e p + N 2 H S

2 +y 1 2 + N 2 H 2 S

2 +P e p + N 2 S 2

2 +P e p + N 2 H 2 S

2 +y 1 2 + N 2 H S 2

2 +P e p + N 2 H 2 S 2

2 +y 1 2 + N 2 H 2 S 2 +

y 1 2 + N 2 H 2 S 2

2 +P e p + N 2 H S 2

2 +P e p + N 2 H S 3

2 +P e p + N 2 H 2 S 3

A

C

B

1 +P e p

Identification of O-Linked Glycopeptides

FT ICR MS/ECD of Glycoproteins

Quantitation of Glycoproteins

Quantitation

Hiroyuki Kaji et al Nature Biotech 2003,(6) 667-672

 Hui Zhang et al, Nature Biotech 2003 (6) pp 660 - 666

1) Trypsin and wash

2) Succinic anhydride

3)

d0 or d4

Mix samples

Quantitative analysis of succinyl peptides (labeled vs unlabled)

4) PNGase F

Biological Mass Spectrometry

Many different MS tools now available to perform all sorts of more and more complex biochemical experiments.

The MS tools as well as software and separation techniques (eg HPLC-Chips) are improving at a very rapid pace. The combinationof these new tools with classical biochemical approaches are especially powerful.

In recent years major improvement in sensitivity, resolution, mass accuracy, throughput, etc.. However some instruments are better at certain tasks than others (ie peptides vs intact proteins,PTMs vs quantitation).