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SC COBRE in Oxidants, Redox Balance, and Stress Signaling

Mass Spectrometry Core

Services Offered: 

The Mass Spectrometry Facility and REDOX Proteomics Core, within the Medical University of South Carolina Proteomics Center provides expertise, services, education and training to enhance biomedical research endeavors through the mass spectrometry-based proteomics. Protein analysis includes proteolytic digestion, chromatographic separation and tandem mass spectrometric analysis (LC-MS/MS), and interpretation of tandem mass spectra using protein database-searching algorithms. The facility assists in the development of customized applications for affinity chromatography and characterization of post-translationally modified peptides (e.g. S/T/Y phosphorylation, S/T O-GlcNAc modification, Cys oxidation and S-glutathionylation, N- and O-linked glycosylation, Lys acetylation, ubiquitylation, and non-enzymatic glycation).  Quantitative proteomic approaches using stable isotope labeling (SILAC), label free proteomics (MaxLFQ, Skyline), or isobaric tagging (iTRAQ, ICAT, TMT) are available to identify differentially expressed proteins and/or differentially regulated sites of post-translational modification from cell lines or tissue samples.

Equipment:

Instrumentation maintained and operated by the Mass Spectrometry Facility, include a hybrid LC-MS/MS Orbitrap Elite Mass Spectrometer (ThermoScientific) with a dual pressure ion trap mass spectrometer for rapid sequencing of peptides and an orbitrap mass analyzer for high mass accuracy measurements. The instrument has multiple peptide fragmentation capabilities including higher energy collisional dissociation (HCD), collision-induced dissociation (CID), and electron transfer dissociation (ETD) for the characterization of complex and fragile post-translational modifications.  The ability to perform MSn with multiple rounds of sequencing and multiple fragmentation modes enables the development of post-translational modification-specific parameters. The instrument is interfaced to a Dionex UltiMate U3000 nanoLC system with an autosampler and 2D-LC capability.

Computational Proteomics Resources and Data Archiving:

Database searching is accomplished with Mascot (v. 2.4 via an in-house 24 CPU 64 GB RAM cluster), MaxQuant (via a 24 core 48 GB RAM high performance server), Byonic, SEQUEST HT (Proteome Discoverer 2.1), Protein Prospector, and X!Tandem (via an in-house 6 CPU cluster).  All workflows incorporate additional search algorithms and tools as needed (e.g., site assignment of post-translational modifications, modification site motif analysis, disulfide linkages).  Results from multiple search algorithms can be combined and/or post-processed using Skyline, Scaffold, Perseus or with built-in tools (i.e., percolator, PhosphoRS) within Proteome Discoverer.  Analyses of SILAC-based experiments for differential expression or post-translational modification are performed with MaxQuant/Perseus; label free analysis of differentially modified proteins with spiked internal standards is performed with Skyline/MStats; isobaric labeling experiments (TMT, SILAC, iTRAQ) are analyzed using Proteome Discoverer. Statistical analysis (both parametric and non-parametric) can be performed within most of these programs, as well as custom R, SigmaPlot, and Matlab. Data are stored locally on high-speed NAS servers and facility file-shares attached to processing computers via gigabit interfaces, and concurrent iterative off-site tape storage is provided through facility TSM storage.  The Mass Spectrometry Facility and REDOX Proteomics Core shares a license for Ingenuity Pathway Analysis software with the MUSC Genomics Center.

The facility provides methodology to analyze alterations in posttranslational regulation that impact signal transduction, protein interactions, epigenetic modulation, and the response to therapeutics, with the goal of enabling investigators to discover molecular mechanisms underlying disease progression and therapeutic response.

Lauren Ball, Ph.D.
Core Director

Associate Professor

Cell and Molecular Pharmacology and Experimental Therapeutics

Jennifer Bethard, M.S.
Core Manager

Mass Spectrometry Facility Manager

Cell and Molecular Pharmacology and Experimental Therapeutics

 
 
 

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