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 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

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

Immunodepletion Allows Lower Abundance Proteins to be identified 10 15 20 25 30 40 50 220 160 120 80 60 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 depletion R2: Rabbit column flow through R3: Rabbit column retentate H1: Human before depletion H2: Human column flow through H3: Human column retentate

Sampling a Gel for Protein Identification 14 13 15 16 12 11 17 10 18 9 19 8 20 7 21 6 22 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 y1 R1 O R2 O R3 O NH3 C C N C C N C C OH b1 b2

MS/MS Spectra Provide Protein Identification

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

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 89 126 409 374 108 70 Gel Gel MuDPIT MuDPIT B) Brain (624) A) Liver (552)

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

Proteomics Core Utilization Grows

The Proteomics Core Serves the University

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