1. The Proteomics Core at Wayne State University
Paul M. Stemmer, Ph.D. Core Director
Joseph A. Caruso, Ph.D. Associate Core Director
Stanley R. Terlecky, Ph.D. Associate Core Director

2. The Proteomics Laboratory

3. Primary Services Offered
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.

4. 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.

5. + - + Investigator Need Drives Proteomics RNA Does the work
Easily quantitated
Easily amplified
RNA +
Analytical Accessibility
Protein +
Does the work -
Impossible to amplify
Difficult to identify
Subject to change
Rarely work in isolation

6. Protein Fragmentation
Proteomics Work Flow
Peptide Mass Analysis
Data Analysis
Protein Separation
Protein Fragmentation

7. Immunodepletion Allows Lower Abundance Proteins to be identified10 15 20 25 30 40 50
220
160
120 80 60
M R1 R2 R 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 depletion
R2: Rabbit column flow through
R3: Rabbit column retentate
H1: Human before depletion
H2: Human column flow through
H3: Human column retentate

8. Sampling a Gel for Protein Identification14 13 15 16 12 11 17 10 18 9 19 8 20 7 21 6 22 5 4 3 2 1

9. Robots for Sample Handing and Processing

10. Mass Spectra Determination on the LTQ-XL

11. Peptides are Fragmented to Generate an MS2 Spectray2 y1 R1 O R2 O R3 O
NH3 C C N C C N C C OH b1 b2

12. MS/MS Spectra Provide Protein Identification

13. Complex Samples MUST be Fractionated Before MS Analysis
Gel Based
MuDPIT
Digest
Ion exchange
Fractionation
Digest

14. Data Analysis and Presentation
MS/MS data is processed through Bioworks 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.

15. 524 Proteins Identified from One Sample

16. Comparison of Gel and MuDPIT Analysis89
126
409
374
108 70
Gel
Gel
MuDPIT
MuDPIT
B) Brain (624)
A) Liver (552)

17. Protein Fragmentation
Proteomics Work Flow
Peptide Mass Analysis
Data Analysis
Protein Separation
Protein Fragmentation

18. Proteomics Core Utilization Grows

19. The Proteomics Core Serves the University

20. Future Plans
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.

21. The People Make It All Work