利用MALDI质谱直接蛋白质交互作用分析

Protein complexes characterization
PEG Protein Characterization Plasma Screening
High-Mass MALDI Imaging
Polymer Analysis
Need a complete slide show for one of these applications? Contact Ryan: ryan.wenzel@
PEBAT SBAT
SEBAT
Worldwide Patents Pending
Thymidine Kinase Dimer Control Disuccinimidyl suberate K100 MALDI Kit
TK TK dimer
TK dimer
High-Mass Detection System
High-Mass Detection Sytem
High Mass Detector OFF MCP detector ON MCP detector HM2 High-Mass Retrofit System High Mass Detector ON MCP detector OFF
•MCP detector remains •Reflectron & MS/MS remain unaffected •Takes about 15 seconds to change positions
HM1 High-Mass Retrofit System
2
High-Mass MALDI ToF System: To detect intact protein complexes up to 1.5 MDa
3
0 500 1000 kDa
Complex Tracker Analysis Software: To evaluate MS data generated
Anti JAK1
Few Examples
Bovine Serum Albumin Anti BSA B2901
Control After Cross-Link
Janus Kinase 1 Anti JAK1 J3774
Control After Cross-Link
Anti BSA [Anti BSA•BSA] 282.13
Proposed Solution
CovalX High Mass Detector
CovalX‘s MALDI protein interaction analysis
1
Cross-linking Kits: To stabilize non-covalent protein complexes for MALDI analysis
Aβ Amyloid Anti Aβ Amyloid BAM403-M
Control After Cross-Link
Anti Aβ Anti Myo 151.78 185.03 [Anti Myo•Myo•Myo] 151.72 161.32 [Anti Aβ• Aβ•Aβ]
16.71
168.48
Bringing MALDI mass spectrometers to direct protein interaction analysis
CovalX AG Grabenstrasse 11a CH-8952 Zürich-Schlieren Switzerland
Method protected by Patents: EU 06 721 947.7, USA 11/919.801, Jap PCTG-3284
Chemical Cross-Linking
Cocktails of Cross-Linkers To increase efficiency
R: Spacer ranging from 6 Å to 16 Å
Two major issues
2) Detection Problem
Standard Detector
•Standard Linear Detectors (MCPs) have known sensitivity limitations in detecting higher mass (slower moving) ions. •MCPs have difficulty detecting saturating with complex solutions where later arriving ions are undetected. •MCPs have higher reaction times and therefore higher resolution when detecting lower mass ions (which require this speed).
Few Examples
Analysis of the intact protein complex formed by HIV-IN and LEDGF
Movable HM2 High-Mass System Independant Electronic and High-Voltage suply
Wireless Control iPod TouchTM CovalX‘s applicatioon Compatibility: • AB SCIEX MALDI TOF/TOF™ 5800 System • AB SCIEX MALDI TOF/TOF™ 4800 System • AB SCIEX MALDI TOF/TOF™ 4700 System • All MALDI TOF Voyager™ Systems
150.47 JAK1
[Anti JAK1•JAK1]
JAK1 BSA
149.72
[Anti BSA•BSA•BSA]
216.92 134.21 284.58 [Anti JAK1•JAK1•JAK1] 66.44 kDa
418.21
Myoglobin Anti Myoglobin MG1
[Anti Myo•Myo] Control After Cross-Link
• Antibody-Antigen interactions • Epitope mapping • Sandwich assays
Fields of Application
Immunoglobulin M Characterization
Inhibitors of Protein-protein interactions Protein Aggregates
147535
Standard detection
223418 299228 292786.2 M ass (m/z) 429181.8 565577.4
33900 31485 0 19995.0
73349 99927 135849 156390.6
Comparison MCP/ High-Mass
173.89 198.71 [6H4•bPrP]
HM2 High-Mass MALDI
6H4
6H4
[6H4•bPrP•bPrP]
149.38nal. Chem.; 2006; 78(11); 3562-3570
Antibody/Antigen interactions Immuno-complex analysis
336.457 kDa 5BSA
Zoom
High-Mass detection
Standard detection
Comparison MCP/ High-Mass
100 146024
IgG 4μM MALDI TOF/TOF™ 4800 System High-Mass detection standard detection
UV laser 337 nm
Flight Tube
Detector AB
UV laser 337 nm
Mass Spectrum
m/z
•Normally ions from a non-covalent complex are dissociated when doing a MALDI experiment •Using chemical stabilization keeps the complex intact so that it can be detected
20
10
0 49913
Zoom
100 90 6573 0 80 67627
70
88391
% I n te n s i ty
60
High-Mass detection
166240 13 4 0 4 7 76 7940
81934
50
40
30
Standard detection
329598 24 6 2 0 3 4960 72 320959 592005 M as s (m/z ) 8630 52 1134098
3IgG
44308 33900 608805 565577.4 701973.0
Zoom
195094 10 21496 48830 99016
High-Mass detection
248111 452009
47349
%I t nsy ne i t
347004 22274
371153 44308 20859 66265 84258 43372 60369 109998 402867
Fight Tube
• Detection beyond 1.5MDa •Low saturation for complex mixtures
MALDI Source
Comparison MCP/ High-Mass
BSA 2μM MALDI TOF/TOF™ 4800 System High-Mass detection Standard detection
Antibody/Antigen interactions Immuno-complex analysis
Few Examples
+ 6H4
149.38
+ bPrP 6H4 Control
+ Cross-link K200 MALDI Kit
HM2 High-Mass MALDI
[6H4•bPrP] [6H4•2bPrP]
20
10 51 2 6 5
0 4991 3
Complex Tracker Protein interaction Analysis Software
Data analysis of the protein interactions data generated by MALDI ToF
Antibody Characterization
Two major issues
1) Dissociation Problem
Source
Complex AB
Flight Tube
Detector B A
Mass Spectrum m/z
Proposed Solution: Chemical Stabilization
Source
Complex AB
100 491983
IgM 1μM MALDI TOF/TOF™ 4800 System High-Mass detection MCP detection
1016.033 kDa 1016.033 kDa
90
80
70
60 % In te n s ity
50
40 323640 30
IgM
1016033 56095 88391 166240 242971 246203 329598 320959 386085 496072 592005 M ass (m /z ) 661438 767940 863052 1134098 1405144
90
80
70
60 % In ten sity
50 296443 40 72784
452.009 kDa
30
20 167903 10 21496 22274 0 19995.0 48830 66265 99016 123087 195094 220984 248111 347004 109998 99927 147535 223418 156390.6 299228 292786.2 Mass (m/z) 429181.8 371153 402867 452009