The Proteomics Core at Wayne State University

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. 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

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

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

7. M R1 R2 R3 H1 H2 H3 220 160 120 80 60 50 40 30 25 20 15 10 Immunodepletion Allows Lower Abundance Proteins to be identified 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 Identification 14 15 13 16 12 17 11 18 10 19 9 8 20 7 21 22 6 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 Spectra y2 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 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.

15. 524 Proteins Identified from One Sample

16. Comparison of Gel and MuDPIT Analysis 108 374 70 409 126 89 Gel MuDPIT Gel MuDPIT B) Brain (624) A) Liver (552)

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

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