The Proteomics Core at Wayne State University Paul M. Stemmer, Ph.D.Core Director Joseph A. Caruso,...
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The Proteomics Core at Wayne The Proteomics Core at Wayne State University State University Paul M. Stemmer, Ph.D. Paul M. Stemmer, Ph.D. Core Director Core Director Joseph A. Caruso, Ph.D. Joseph A. Caruso, Ph.D. Associate Core Associate Core Director Director Stanley R. Terlecky, Ph.D. Stanley R. Terlecky, Ph.D. Associate Core Associate Core Director Director
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Transcript of The Proteomics Core at Wayne State University Paul M. Stemmer, Ph.D.Core Director Joseph A. Caruso,...
The Proteomics Core at Wayne State The Proteomics Core at Wayne State UniversityUniversity
Paul M. Stemmer, Ph.D.Paul M. Stemmer, Ph.D. Core DirectorCore Director
Joseph A. Caruso, Ph.D.Joseph A. Caruso, Ph.D. Associate Core DirectorAssociate Core Director
Stanley R. Terlecky, Ph.D.Stanley R. Terlecky, Ph.D. Associate Core DirectorAssociate Core Director
The Proteomics Laboratory
Services are offered for protein identification, proteome profiling and MS-based relative protein quantitation.
nanoLC/MS/MS using the LTQ-XL Linear Ion Trap with ETD. Triple Quad MS for MRM analysis using a TSQ Voyager. MALDI ToF MS using the ABI Voyager-DE Pro. Proteome profiling by two dimensional chromatography. Proteome fractionation by preparative gel electrophoresis or by
isoelectric focusing using free flow electrophoresis. Serum or plasma depletion of abundant proteins from human or rodent samples. Protease or chemical based fragmentation of proteins in
preparation for mass spectrometry. Data analysis using Sequest, Mascot, Peaks and X!tandem
algorithms. Data interpretation, presentation and publication tools using
Scaffold software.
Primary Services Offered
Proteomics: A Technology Driven Discipline
Technological advances have made MS based protein identification and sequencing possible.
MS based proteomics is dependent upon up-to-date database and search engine capabilities.
Investigator Need Drives ProteomicsInvestigator Need Drives Proteomics
Protein
Does the workDoes the work+
• Impossible to amplifyImpossible to amplify
• Difficult to identifyDifficult to identify
• Subject to changeSubject to change
• Rarely work in isolationRarely work in isolation
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• Easily quantitatedEasily quantitated
• Easily amplifiedEasily amplified
RNA
+ Analytical Analytical AccessibilityAccessibility
Proteomics Work Flow
Protein Separation
Protein Fragmentation
Peptide Mass Analysis
Data Analysis
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M R1 R2 R3 H1 H2 H3
Rabbit (200µl) or human plasma (250 µl) were depleted of 12 abundant proteins by a single pass over a IgY-12 column (Beckman Coulter) designed for depletion of human serum and plasma. SDS-PAGE with coomassie blue staining is shown. 20 µg protein was applied to each lane.
Samples are: R1: Rabbit before depletionR2: Rabbit column flow throughR3: Rabbit column retentateH1: Human before depletionH2: Human column flow throughH3: Human column retentate
Immunodepletion Allows Lower Abundance Proteins to be identified
Sampling a Gel for Protein Sampling a Gel for Protein IdentificationIdentification
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Robots for Sample Handing and ProcessingRobots for Sample Handing and Processing
Mass Spectra Determination on the LTQ-XL
C C N CNH3 C
R1 R2O O
N C C
R3 O
OH
b1 b2
y1y2
Peptides are Fragmented to Generate an MS2 Spectra
MS/MS Spectra Provide Protein Identification
Complex Samples MUST be Fractionated Before MS Analysis
C:\XCalibur\...\Raw\MuDPIT\11-26-07_30 11/26/2007 10:10:17 PM
RT: 0.00 - 45.00
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Time (min)
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NL:1.57E7
Base Peak F: ITMS + c NSI Full ms [300.00-2000.00] MS 11-26-07_30
11-26-07_30 #3110 RT: 16.76 AV: 1 NL: 5.49E3T: ITMS + c NSI d Full ms2 [email protected] [265.00-2000.00]
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m/z
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433.21 1507.381194.32832.33 1793.58562.34
1557.40332.25 1065.27402.28 1658.33667.17501.11 778.39 1861.521769.481428.281288.41 1911.97
Digest
Digest
Ion exchangeFractionation
MuDPITGel Based
Data Analysis and Presentation
MS/MS data is processed through Bioworks 3.3.1 using the Sequest algorithm.
Processed data is analyzed with Scaffold software using the X! tandem algorithm.
Data from both Sequest and X! tandem analyses are presented in Scaffold.
524 Proteins Identified from One Sample
Comparison of Gel and MuDPIT Analysis
A) Liver (552)B) Brain (624)
374 1087089 126409
MuDPITGel Gel MuDPIT
Proteomics Work Flow
Protein Separation
Protein Fragmentation
Peptide Mass Analysis
Data Analysis
2006 2007 20080
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Consultation
Analysis of Sample
Invest
igato
rs U
tilizi
ng t
he P
rote
om
ics
Core
Proteomics Core Utilization Grows
IEHS, 11 Projects
School of Medicine, 8 Projects
Outside the University, 7 Projects
Karmanos Cancer Inst., 9 Projects
WSU Main Campus, 5 Projects
The Proteomics Core Serves the University
Initiate service using isobaric tags for differential proteomic analysis.Establish efficient work flows for identification of post translational modifications on proteins using the ETD feature of the LTQ-XL.Obtain instrumentation for Selective Ion Monitoring (SIM) and Multiple Reaction Monitoring (MRM) to validate peptide biomarkers.
Future Plans
The People Make It All Work
