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 proteinsfrom 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 quantitatedEasily amplifiedRNA+Analytical AccessibilityProtein+Does the work-Impossible to amplifyDifficult to identifySubject to changeRarely work in isolation
6 Protein Fragmentation Proteomics Work FlowPeptide Mass AnalysisData AnalysisProtein SeparationProtein Fragmentation
7 Immunodepletion Allows Lower Abundance Proteins to be identified 101520253040502201601208060M R1 R2 R H1 H2 H3Rabbit (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
8 Sampling a Gel for Protein Identification 14131516121117101891982072162254321
13 Complex Samples MUST be Fractionated Before MS Analysis Gel BasedMuDPITDigestIon exchangeFractionationDigest
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.
20 Future PlansInitiate 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.