Protein Cross-Linking Analysis using
Stable Isotope-Labeling, Mass Spectrometry,
and Integrated Computational Data Processing
Jan Seebacher
Aebersold Lab, ISB, Seattle WA
In Collaboration with Michael Gelb, University of Washington
2
3D Structure Determination of Proteins and Protein Complexes
Molecular Biology /
Affinity Purification /
Mass Spectrometry
NMR
B A
C
Sequence
Protein Complex /
Protein Interactions
?
Candidate Structures
Protein Crystallography
3D Protein Structure Prediction
Modeling
3
3D Structure Determination of Proteins and Protein Complexes
Chemical Cross-Linking
Constraints
Molecular Biology /
Affinity Purification /
Mass Spectrometry
NMR
B A
C
Sequence
Protein Complex /
Protein Interactions
?
Candidate Structures
Protein Crystallography
3D Protein Structure Prediction
Modeling
4
Protein Cross-linking to Study 3D Protein Structure: Proof of Concept
3D Protein Structure
A
B
Protein Complex
(Proteins A+B)
known 3D structure, pdb.
Method Validation
“
”
Cross-Linking Reagent
“Molecular Ruler”
Cross-linking Reaction
Protein Sequence A
Protein Sequence B
Identification of Cross-linked AA Residues (K, n)
Æ Inter-Residue Distance Constraints
Æ Protein Structure Prediction
Æ Detection of Protein Interactions / Sites
5
Method Outline: Exp. Workflow + Data Analysis
1:1 Ratio
• Peptide Identification
18
O-Isotope-Coded Buffer
[d0/d12]-isotope-coded
Cross-Linker
[16O]
• Identification of Cross-Linked Residues
[16/18O]
• Generation of “Distance Constraints” for 3D
Structure Modeling, i.e. for Rosetta, NMR, etc.
• Identification of potential Interaction Sites
1
+
Automated MS/MS Analysis
2 1
[16O]
Protein Cross-linking
2
Automated MS Analysis:
Digestion, :
Proteins Æ Peptides
MS2 (MS/MS) Acquisition
Robotic LC Fractionation
onto MALDI Plate
• Isotope pattern recognition
• Spectra Alignment
• Peptide Data Base Search
• Mass Mapping Æ Candidates
• Æ MS2 Inclusion List
[16O]
MS1 Acquisition
ABI 4700 Proteomics Analyzer
[16/18O]
6
Strategy: Protein Cross-Linking and MS Analysis
Protein-reactive Group
A
B
+
A
B
Cross-Linking Reagent
dead-end, hydrolyzed
Æ Mono-Link
Protease(s)
Peptides
Æ Identification by
Mass Spectrometry
intra-complex
Cross-Links
Protein Complex
Æ Digestion Products:
Unmodified Peptides + Mono-Links
Loop-Link
Cross-Links
7
MS1 Analysis of isotope-coded cross-linked peptides
Cross-linking Reagent
Unmodified Peptides + Mono-Links
Cross-Links
Loop-Link
H
XX
XX
O
XX
Peptide
Peptide N
Peptide
N
O
XX
XX
XX
XX
H
XX
N
O
[16/18O]H
O
H
XX
XX
XX
XX
X: 50% H/D
[16O]
12 Da
2 Da
[16/18O]
Æ Cross-Link or Loop-Link
Æ Mono-Link
8
MS1 Analysis of isotope-coded cross-linked peptides
Cross-linking Reagent
Unmodified Peptides + Mono-Links
Cross-Links
Loop-Link
H
XX
XX
O
XX
Peptide
Peptide N
Peptide
N
O
XX
XX
XX
XX
H
XX
N
O
[16/18O]H
O
H
XX
XX
XX
XX
X: 50% H/D
[16O]
12 Da
2 Da
[16/18O]
This can be automated Æ development of Software Tools
9
MS1 Data Analysis with iXLINK (Parag Mallick)
Example MS1 spectra - d0/d12-DSS
4700 Reflector Spec #1 MC[BP = 2302.2, 7632]
100
90
7631.6
O
O
O
O
N
N
O
80
O
2301.1851
O
O
70
% Intensity
60
2313.2556
50
12 Da
40
30
2358.2075
20
2370.2810
2230.1462
10
2312.2522
2242.2180
2287.1707
2241.2261
0
2205
2241
2284.0977
2299.2451
2297.0779
2277
2311.2649
2313
2327.1445
2344.1980
2349
2356.2422
2369.2783
2385
Mass (m/z)
ABI 4700 Proteomics Analyzer
Example MS1 spectra - d0/d6-DSG
4700 Reflector Spec #1 MC[BP = 2260.1, 2788]
100
2787.8
90
O
O
O
O
N
80
2259.1016
N
O
O
O
O
70
6 Da
% Intensity
60
50
2265.1360
2188.0684
40
30
2194.1018
20
2316.1274
2322.1523
2245.0886
10
2332.1814
2251.1289
2174.0688
0
2157.0
2193.0959
2231.0933
2198.6
2250.1165
2240.2
2287.1348
2281.8
2303.1057
2331.1892
2323.4
2365.0
Mass (m/z)
ABI 4700 Proteomics Analyzer
Example MS1 spectra – d0/d4-BS3
4700 Reflector S pec #1 MC[BP = 2304.4, 2244]
100
2243.7
For comparison:
2303.4473
90
80
70
% Intensity
60
2299.4331
50
40
4 Da
30
2360.3914
20
2232.5237
2228.5054
2356.3596
10
2289.4583
2285.4299
2223.4497
0
2208.0
2325.3496
2324.3579
2278.3958
2245.2
2282.4
2319.6
2356.8
2394.0
M ass (m/z)
ABI 4700 Proteomics Analyzer
13
Example LC- MALDI MS1 Data (iXLINK output)
Æ Isotope Patterns
3400
12
3300
3200
m/z [Da]
3100
1.85
1.90
1.95
LC-MS Fraction (“Retention Time”)
[16O] - Peptide Sample
[16/18O] - Peptide Sample Image created with program “MTPeak” by Paul Loriaux
14
MS Analysis Workflow
12 Da
* *
*
*
d0
doXLINK Analysis (Ning Zhang)
m/z
d0
m/z
d12
d12
Mass Pairs
m/z
MS2 - Acquisition
- MS2 Spectra Alignment
- Isotope Pattern (Reporter Ions)
- iXLINK output (Peptide Candidates)
- Peptide Fragment Ion Mass Matching
- Peptide Scoring (ProbID[1])
iXLINK Inclusion List
User Validation
[1] N. Zhang et al. (2002) Proteomics 2(10): 1406-1412
15
A “good” MALDI MS/MS spectrum of a light/heavy cross-linked peptide pair
*
*
d0
*
*
*
d12
ABI 4700 Proteomics Analyzer
16
A “good” MALDI MS/MS spectrum of a light/heavy cross-linked peptide pair
*
+ 12 Da
Reporter Ion
*
*
d0
*
Reporter Ion
d12
*
*
17
Some typical MALDI MS/MS spectra of cross-linker- modified peptide species
Reporter Ions
Cross-Link
m/z = 222.1
Mono-Link
m/z = 240.1
18
Validation of Computed Cross-Linking Results with XLinkViewer (James Eddes)
19
Analysis Results
Matched Peptides
Error [Da/ppm]
“Score”
Locations of cross-linked
Residues in Protein-Complex
User-Validation
20
Final Results – 3D Structure (1ujz.pdb) – two cross-linking reagents
O
O
O
O
N
N
O
O
DSG (7.7 Å)
O
O
O
O
O
O
N
N
O
O
O
O
DSS (11.4 Å)
Colicin E7 DNAse/Im7 protein complex [2]
[2] Kortemme, T. et al. Nat. Struct. Mol. Biol. 2004, 11, 371-379.
21
Conclusions from this Study
Æ Protein cross-linking protocol was developed using isotope-labeling and LC-MALDI
mass spectrometry
Æ Three integrative software tools were developed to analyze protein cross-linking MS
and MS/MS data: iXLINK, doXLINK and XLinkViewer
Æ All software is available for download from the SPC website
Æ 23 cross-linked amino acid residues were identified in the Colicin E7 DNAse/Im7
Protein complex (with d0/12-DSS and d0/d6-DSG cross-linking reagents)
Æ all lysine residues within < 22.1 Å distance of each other Æ distance constraints
(as expected from the cross-linker chain length)
Æ 5-10 ug of protein was used per experiment
Æ Compatible with various common isotope-coded bis-NHS ester cross-linking reagents
(readily synthesized, or i.e. [d0/d4]-BS2G and BS3 from Pierce)
Æ Method has been validated with additional 5 single proteins (1 week analysis time)
Æ Experiment + analysis can be automated
Æ Potential for high-throughput protein structure analysis
22
References
Web-Links:
http://www.proteomecenter.org/ASMS/Seebacher_ASMS_06_Poster.pdf
http://tools.proteomecenter.org/XLink.php
____________2006 ASMS poster
____download software + manual
Original Publication:
Seebacher, J., Mallick, P., Zhang, N., Eddes, J. S., Aebersold, R., Gelb, M. H.
"Protein Cross-linking Analysis Using Mass Spectrometry, Isotope-Coded Cross-linkers, and Integrative Computational Data
Processing"
J. Proteome Res., 2006; 5: 2270-2282.
http://www.piercenet.com
Acknowledgements
University of Washington
• Parag Mallick (Cedars -Sinai)
• Ning Zhang
• James S. Eddes
• Nichole King
• Richard Bonneau (NYU)
• Simon Letarte
• Sheng Pan
• Bernd Wollscheid (IMSB)
• Ruedi Aebersold
• NHLBI, SPC
• Lars Malmström
• David Baker
• Brian Smart (UCB)
• Michael Gelb