Transcript Slide 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 The Proteomics Laboratory 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. 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 Proteomics Protein RNA + + Does the work • Impossible to amplify Analytical Accessibility • Difficult to identify • Easily quantitated • Subject to change • Easily amplified • Rarely work in isolation - Proteomics Work Flow Protein Separation Protein Fragmentation Peptide Mass Analysis Data Analysis Immunodepletion Allows Lower Abundance Proteins to be identified M 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 220 160 120 80 60 50 40 30 25 20 15 10 R1 R2 R3 H1 H2 H3 Sampling a Gel for Protein Identification 14 11 10 8 7 15 16 17 18 19 20 21 6 22 13 12 9 5 4 3 2 1 Robots for Sample Handing and Processing Mass Spectra Determination on the LTQ-XL Peptides are Fragmented to Generate an MS2 Spectra y2 NH3 R1 O C C b1 N y1 R2 O C C b2 N R3 O C C OH MS/MS Spectra Provide Protein Identification Complex Samples MUST be Fractionated Before MS Analysis Gel Based MuDPIT Digest Ion exchange Fractionation C:\XCalibur\...\Raw\MuDPIT\11-26-07_30 11/26/2007 10:10:17 PM RT: 0.00 - 45.00 100 NL: 1.57E7 Bas e Peak F: ITMS + c NSI Full m s [300.002000.00] MS 11-26-07_30 90 80 Relative Abundance 70 60 50 40 30 20 10 0 0 2 4 6 8 10 12 14 16 18 11-26-07_30 #3110 RT: 16.76 AV: 1 NL: 5.49E3 T: ITMS + c NSI d Full m s 2 [email protected] [265.00-2000.00] 100 90 80 897.92 20 22 24 Tim e (m in) 26 28 30 32 34 36 38 40 42 44 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 Gel 70 MuDPIT 374 108 A) Liver (552) Gel 89 MuDPIT 409 126 B) Brain (624) Proteomics Work Flow Protein Separation Protein Fragmentation Peptide Mass Analysis Data Analysis Proteomics Core Utilization Grows 25 Consultation Investigators Utilizing the Proteomics Core 20 Analysis of Sample 15 10 5 0 2006 2007 2008 The Proteomics Core Serves the University WSU Main Campus, 5 Projects Karmanos Cancer Inst., 9 Projects Outside the University, 7 Projects School of Medicine, 8 Projects IEHS, 11 Projects 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. The People Make It All Work