Biomolecule analysis with electrospray mass spectrometry
Dr. Harry BrumerWood Biotechnology LaboratoryKTH Department of [email protected]
Molecular Biotechnology Prof Mathias Uhlén Prof Joakim Lundeberg Prof Per-Åke Nygren Prof Stefan Ståhl
Biochemistry Prof Karl Hult Prof Pål Nyrén
Dept. of Biotechnology~200 personer
ca 80 pers
ca 20 pers
Wood Biotechnology Prof Tuula Teeri
ca 25 pers
Bioprocess technology Prof Sven-Olof Enfors Prof Lena Häggström ca
30 pers
Environmental Microbiology Assoc. Prof Gunnel Dalhammar
ca 10 pers
Structural BiochemistryProf Torleif Härd
ca 15 pers
Theoretical ChemistryProf Hans Ågren
Prof Faris Gelmukhanov
ca 15 pers
Adm
ca 5 pers
KTH Department of Biotechnology
http://www.biotech.kth.se/woodbiotechnology/
MS in the Wood Biotechnology Lab
Equipment purchase funded by the Wallenberg Consortium North for Functional Genomics
Micromass Q-TOF2 orthogonal acceleration quadrupole/time-of-flight MS
Orthogonal (Z-spray) electrospray ionisation interface
Nanoflow LC system (200 nL/min)
MS Components
Ionisation source
ion optics, including one or more mass analysers
detector
analyte ionisation mass selection
detection
EI, CI, FAB, MALDI, ESI
mag. sector, quadrupole, TOF, ion trap, FT-ICR
faraday cup, dynode, scintillation, MCP
Hybrid quadrupole-TOF
Hybrid quadrupole-TOF
Hybrid quadrupole-TOF
Electrospray ionisation (ESI)
Biological molecules are often non-volatile and/or thermally labile:
proteins/peptidesnucleic acids (DNA, RNA)carbohydrates
EI and CI not applicable w/o derivitisationFAB mass range limited
OK for small carbohydrates, peptides
MALDI good for biologicalsnot dynamic, typically gives [M+H]+ ions
ESIsuitable for on-line methods (CE, LC)gives multiply charged ions
Electrospray ionisation (ESI)
ESI: Ion production
“Z-spray” ESI source
ESI yields multiply-charged ions
Normal quadrupoles can be used for high-mass biomolecules
Hybrid quadrupole-TOF
Time-of-flight
analyzers (TOF)
K.E. = 1/2 mv2
Orthogonal acceleration TOF (oaTOF)
used in ESI-TOF applications
Hybrid quadrupole-TOF
Quadrupole mass analyzer
Q1 can be tuned to allow only ions of a single m/z to pass
Hybrid quadrupole-TOF
The collision cell
1 pmol/uL in 1:1 MeOH/water, 0.2 % formic 05-Mar-2002 11:09:19
600 800 1000 1200 1400m/z0
100
%
GLUFIB5 47 (2.014) Cm (24:56) TOF MS ES+ 7.62e3785.848
786.339
786.842
Glu-fibrinogen peptide: TOF MS
783 784 785 786 787 788 789 790 791m/z0
100
%
GLUFIB5 47 (2.014) Cm (24:56) TOF MS ES+ 7.62e3785.848
786.339
786.842
0.5 m/z separation of isotopic peaks: [M+2H]2+
Peptide sequencing by MS/MS
Amino acids differ in their side chains
Predominant fragmentation
Weakest bonds
Glu-fib ESI-TOF MS/MS1 pmol/uL, 10ul/min 23-Oct-2001 18:54:19
200 400 600 800 1000 1200 1400 1600 1800m/z0
100
%
GLUFIB_MSMS_23OCT2001 121 (5.362) Cm (87:125) TOF MSMS 785.80ES+ 2.55e3333.188
187.073
684.339
480.252
497.199
813.396
1056.469
1285.511
1286.520
0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000M/z0
100
%
intermediate soln.GLUFIB_NANO_MSMS_19NOV MaxEnt 3 95 [Ev-188973,It50,En1] (0.050,200.00,0.200,1400.00,2,Cmp) 1: TOF MSMS 785.85ES+
R A S F F G E E N D N V G E yMax684.36
y6
480.27y4
333.20y3
187.09b2
175.13y1
169.0772.10V
246.17y2
382.19
497.21
627.35y5
612.24
813.41y7
740.30
1570.72(M+H) +1285.58
y11
1056.52y9942.47
y8
924.431039.46
1171.53y10
1057.44
1268.551535.71
1517.661384.67y12
1515.68
1571.86
1588.94
Raw data
MaxEnt 3 deisotoped, deconvoluted data
Glu-Gly-Val-Asn-Asp-Asn-Glu-Glu-Gly-Phe-Ph-Ser-Ala-Arg
31-Oct-2001 15:00:03
500 600 700 800 900 1000 1100 1200 1300 1400m/z0
100
%
BOV_INSULIN 148 (6.527) Cm (148:176) TOF MS ES+ 4.05e4956.3642
956.8810
1147.4683
ESI-TOF MS of bovine insulin, 5736 Da
955 956 957 958m/z0
100
%
BOV_INSULIN 148 (6.527) Cm (148:176) TOF MS ES+ 4.05e4956.3642
956.2003
956.0491
956.5406
956.7046
956.8810
Isotopic peaks sep’d. by 0.16 m/z -> (M+6H)6+
horse heart myoglobin 24-Oct-2001 17:05:29
600 800 1000 1200 1400 1600 1800m/z0
100
%
HHM 1 (0.094) Cm (1:32) TOF MS ES+ 7.62e3616.1976
942.7773617.1996 1060.4655
1131.0928
1304.9095
Horse heart myoglobin (HHM)
ESI-TOF MS
1056 1058 1060 1062 1064 1066 1068m/z0
100
%
HHM 1 (0.094) Cm (1:32) TOF MS ES+ 3.26e31060.4655
1061.7930 1063.0416
1065.5413
No charge state information available - insufficient resolution
HHM - deconvoluted masshorse heart myoglobin 24-Oct-2001 17:05:29
10000 12000 14000 16000 18000mass0
100
%
HHM 10 (0.869) M1 [Ev-176947,It11] (Gs,0.400,751:1961,1.00,L33,R33); Cm (4:21)6.89e416951.176
From system of equations: [M+n(H+)]/n
MaxEnt 1
Charge state distributions
600 800 1000 1200 1400 1600 1800m/z0
100
%
HHM 1 (0.094) Cm (1:32) TOF MS ES+ 7.62e3616.1976
942.7773617.1996 1060.4655
1131.0928
1304.9095
(M+21H)21+
(M+9H)9+
in ACN/water + 1% formic 19-Nov-2001 15:29:30
600 800 1000 1200 1400 1600 1800m/z0
100
%
YADH_NANO_19NOV 15 (0.673) Cm (13:44) TOF MS ES+ 1.52e31149.3616
1050.9436
855.6663
799.9018
1268.1848
1362.0247
1414.3512
1470.913936749.4 Da
ca. 20+ to 50+
16952 Da
ca. 9+ to 21+
Monitoring deglycosylation of XET
Intact protein mass analysis of a glycoprotein
Glycosylation removed enzymatically
Kinetics followed by ESI-MS
Extent of deglycosylation can be correlated w/ changes in protein function
LC-MS & LC-MS/MS
LC-MS/MS for glycopeptide discovery
Reconstructed chromatogram showing only sugar signature ions (m/z 163, 204, & 366)
TOF MS “survey” total ion chromatogram
MS/MS spectra acquired in real time
Detailed MS/MS analysis
Localise site of glycan attachment in the protein
Comparison of MS/MS sequence information
with other XETs
Allows us to define the site of glycosylation for the entire protein family
Henriksson, et al. (2003) Biochemical Journal, 375, 61-73.
Oligosaccharide MS/MS analysis: Borriss, R., et al. (2003) Carbohydrate Research, 338, 1455-1467.
ArabidopsisTCH4 SAGTVTTLYLKSPGTTWDEIDFEFLGNS SGEPYTLHTNVYTQGKGDKEQQFKLWFDPTANFH 140XET1 SAGTVTAFYLSSQNSEHDEIDFEFLGNR TGQPYILQTNVFTGGKGDREQRIYLWFDPTKEFH 150Kiwi SAGTVTAFYLSSQNSEHDEIDFEFLGNR TGQPYILQTNVFTGGKGDREQRIYLWFDPTKDYH 142Tomato SAGVVTAFYLSSNNAEHDEIDFEFLGNR TGQPYILQTNVFTGGKGNREQRIYLWFDPTKGYH 150Tobacco SAGVVTAFYLSSNNAEHDEIDFEFLGNR TGQPYILQTNVFTGGKGDREQRIYLWFDPTKGYH 149Soybean SAGTVTAFYLSSQNAEHDEIDFEFLGNR TGQPYILQTNVFTGGKGDREQRIYLWFDPTKEYH 147MS/MS seq. YLSSTNNEHDELDFEFLGDR TGQPVLLQTNVFTGGK ***.**::**.* **********. :*:** *:***:* ***::**:: ****** :*