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Protein Cross-Linking Analysis using Stable Isotope...
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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
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Transcript of Protein Cross-Linking Analysis using Stable Isotope...
Protein Cross-Linking Analysis using Stable Isotope-Labeling, Mass Spectrometry,
and Integrated Computational Data Processing
Jan Seebacher
Aebersold Lab, ISB, Seattle WAIn Collaboration with Michael Gelb, University of Washington
3D Structure Determination of Proteins and Protein Complexes2
B A
C
NMR
Protein Crystallography
Protein Complex /Protein Interactions
Sequence
Candidate Structures
3D Protein Structure Prediction
Molecular Biology /Affinity Purification /Mass Spectrometry
Modeling
??
3D Structure Determination of Proteins and Protein Complexes3
B
NMR
Protein Crystallography
Protein Complex /Protein Interactions
Sequence
Candidate Structures
3D Protein Structure Prediction
Molecular Biology /Affinity Purification /Mass Spectrometry
Modeling
Constraints
Chemical Cross-Linking
?
A
C
Protein Complex(Proteins A+B)
known 3D structure, pdb.
Method Validation
Protein Sequence A
Protein Sequence B
Cross-Linking Reagent“Molecular Ruler”
“ ”
Inter-Residue Distance ConstraintsProtein Structure PredictionDetection of Protein Interactions / Sites
Cross-linking Reaction
Identification of Cross-linked AA Residues (K, n)
3D Protein Structure
Protein Cross-linking to Study 3D Protein Structure: Proof of Concept4
BA
Digestion, :Proteins Peptides
Protein Cross-linking
[16/18O][16O]
+
[d0/d12]-isotope-codedCross-Linker
1
2
2 1
ABI 4700 Proteomics Analyzer
[16/18O][16O]Robotic LC Fractionation
onto MALDI Plate
Automated MS Analysis:• Isotope pattern recognition• Spectra Alignment• Peptide Data Base Search • Mass Mapping Candidates• MS2 Inclusion List
Automated MS/MS Analysis
• Peptide Identification
• Identification of Cross-Linked Residues
• Generation of “Distance Constraints” for 3D Structure Modeling, i.e. for Rosetta, NMR, etc.
• Identification of potential Interaction Sites
MS1 Acquisition
MS2 (MS/MS) Acquisition
[16O]
18O-Isotope-Coded Buffer
Method Outline: Exp. Workflow + Data Analysis5
1:1 Ratio
Cross-Linking Reagent
Unmodified Peptides + Mono-Links Cross-Links
Protein Complex
Protein-reactive Group
Loop-Link
+
BA
Peptides
Identification by Mass Spectrometry
Digestion Products:
Protease(s)BA
intra-complexCross-Links
dead-end, hydrolyzedMono-Link
6Strategy: Protein Cross-Linking and MS Analysis
Mono-LinkCross-Link or Loop-Link
Peptide N N
O
X X
X X
X X
X X
X X
X X
OH
H
Peptide
2 Da
N
O
X X
X X
X X
X X
X X
X X
OHPeptide [16/18O]H
X: 50% H/D
12 Da
7
[16/18O]
[16O]
Unmodified Peptides + Mono-LinksCross-LinksLoop-Link
MS1 Analysis of isotope-coded cross-linked peptides
Cross-linking Reagent
Peptide N N
O
X X
X X
X X
X X
X X
X X
OH
H
Peptide
[16/18O]
[16O]
2 Da
N
O
X X
X X
X X
X X
X X
X X
OHPeptide [16/18O]H
X: 50% H/D
12 Da
8
Unmodified Peptides + Mono-LinksCross-LinksLoop-Link
MS1 Analysis of isotope-coded cross-linked peptides
This can be automated development of Software Tools
Cross-linking Reagent
MS1 Data Analysis with iXLINK (Parag Mallick)9
2205 2241 2277 2313 2349 2385Mass (m/z)
7631.6
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
4700 Reflector Spec #1 MC[BP = 2302.2, 7632]
2301.1851
2313.2556
2358.2075
2370.28102230.1462
2312.25222242.2180
2287.17072299.2451
2369.27832241.2261 2327.14452311.26492297.07792284.0977 2344.1980 2356.2422
Example MS1 spectra - d0/d12-DSS
12 Da
ABI 4700 Proteomics Analyzer
N
O
O
O
O
ON
O
O
O
2157.0 2198.6 2240.2 2281.8 2323.4 2365.0Mass (m/z)
2787.8
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
4700 Reflector Spec #1 MC[BP = 2260.1, 2788]
2259.1016
2265.13602188.0684
2194.1018
2316.1274
2322.15232245.0886
2332.18142251.12892193.0959 2303.10572250.11652174.0688 2331.18922231.0933 2287.1348
Example MS1 spectra - d0/d6-DSG
ON
O
O
O
ON
O
O
O
6 Da
ABI 4700 Proteomics Analyzer
2208.0 2245.2 2282.4 2319.6 2356.8 2394.0Mass (m/z)
2243.7
0
10
20
30
40
50
60
70
80
90
100
% I
nten
sity
4700 Reflector Spec #1 MC[BP = 2304.4, 2244]
2303.4473
2299.4331
2360.3914
2232.52372228.5054 2356.3596
2289.45832285.42992325.34962324.35792223.4497 2278.3958
Example MS1 spectra – d0/d4-BS3
4 Da
ABI 4700 Proteomics Analyzer
For comparison:
m/z
[Da]
1.85 1.90 1.95
3100
3200
3300
3400 12
[16O] - Peptide Sample[16/18O] - Peptide Sample Image created with program “MTPeak” by Paul Loriaux
Example LC- MALDI MS1 Data (iXLINK output)
LC-MS Fraction (“Retention Time”)
13
Isotope Patterns
MS2 - Acquisition
MS Analysis Workflow
- MS2 Spectra Alignment- Isotope Pattern (Reporter Ions)- iXLINK output (Peptide Candidates)- Peptide Fragment Ion Mass Matching- Peptide Scoring (ProbID[1])
doXLINK Analysis (Ning Zhang)
Mass Pairs
User Validation
14
12 Da
d0 d12
iXLINK Inclusion List
d0
d12m/z
m/z
m/z
[1] N. Zhang et al. (2002) Proteomics 2(10): 1406-1412
* **
*
A “good” MALDI MS/MS spectrum of a light/heavy cross-linked peptide pair
**
*
* *
15
d0
d12
ABI 4700 Proteomics Analyzer
**
*
* *
A “good” MALDI MS/MS spectrum of a light/heavy cross-linked peptide pair
Reporter Ion
Reporter Ion
+ 12 Da
16
d0
d12
**
*
**
*
Some typical MALDI MS/MS spectra of cross-linker- modified peptide species
Mono-Link
Cross-Link
Reporter Ions
17
m/z = 222.1
m/z = 240.1
18Validation of Computed Cross-Linking Results with XLinkViewer (James Eddes)
User-ValidationLocations of cross-linkedResidues in Protein-Complex
Error [Da/ppm]
Matched Peptides “Score”
Analysis Results19
[2] Kortemme, T. et al. Nat. Struct. Mol. Biol. 2004, 11, 371-379.
Colicin E7 DNAse/Im7 protein complex [2]
Final Results – 3D Structure (1ujz.pdb) – two cross-linking reagents20
ON
O
O
O
ON
O
O
O
N
O
O
O
O
ON
O
O
O
DSG (7.7 Å)
DSS (11.4 Å)
Protein cross-linking protocol was developed using isotope-labeling and LC-MALDI mass spectrometry
Three integrative software tools were developed to analyze protein cross-linking MS and MS/MS data: iXLINK, doXLINK and XLinkViewer
All software is available for download from the SPC website
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)
all lysine residues within < 22.1 Å distance of each other distance constraints(as expected from the cross-linker chain length)
5-10 ug of protein was used per experiment
Compatible with various common isotope-coded bis-NHS ester cross-linking reagents (readily synthesized, or i.e. [d0/d4]-BS2G and BS3 from Pierce)
Method has been validated with additional 5 single proteins (1 week analysis time)
Experiment + analysis can be automated
Potential for high-throughput protein structure analysis
21Conclusions from this Study
Web-Links:http://www.proteomecenter.org/ASMS/Seebacher_ASMS_06_Poster.pdf ____________2006 ASMS posterhttp://tools.proteomecenter.org/XLink.php ____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
22References
• 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
University of Washington
Acknowledgements
