The Proteomics Shared Resources (PSR) is an interdisciplinary unit that supports high-quality cancer research by providing technical expertise and state-of-the-art instrumentation to the OSUCCC-James and the Ohio cancer research community.
The PSR supports standard proteomic assays and provides innovative collaborative research opportunities to enhance cancer-relevant studies. It offers cancer investigators the critical tools and expertise needed for identifying proteins, quantifying protein expression levels, discovery of protein modifications and protein biomarkers.
The resource offers cell lysis of cell pellets or protein extraction from cells, tissue or other biological media, protein quantitation, and can assist researchers in optimizing protein purification. Following protein preparation from cells or tissue, protein separation services using western blotting, 1D or 2D-PAGE techniques are available. The gels can be stained with various stains including Coommassie, Sypro Ruby, Deep Purple and Silver. Multiplex analysis by visualization of proteins and protein modifications using specialized fluorescent stains, and advanced gel image analysis are available to study phosphorylation, glycosylation and other modifications. Changes in protein expression levels are analyzed using Differential Image Gel Electrophoresis (DIGE) which uses cy dye labeling. Recently the PSR has developed and implemented iTRAQ technology using the new LTQ-Orbitrap. iTRAQ (isobaric tag for relative and absolute quantitation) is a non-gel based technique used to identify and quantify proteins/peptides from different sources in one single experiment by using isotope coded covalent tags that will label the N-terminus and side chain amines of peptides from protein digestions.
Protein spots of interest can be determined following excision from the polyacrylamide gel, protease digestion, and nano LC-MS/MS analysis of the peptides on either a LTQ or LTQ Orbitrap mass spectrometer. Nano-LC/MS/MS is used to provide internal sequence of the protein and has many advantages over traditional MALDI analyses. We have found this method to be sensitive and powerful, producing numerous sequences from low fmole of material and enabling us to identify proteins for which peptide mass mapping by MALDI was unsuccessful. The Capillary LC system is capable of 2-D LC-chromatography for the analysis and identification for complex protein mixtures (global proteomics – MudPIT). The LTQ Orbitrap mass spectrometer allows for high resolution mass analysis of peptides for accurate post-translational modification studies.
The PSR’s goals are to:
1. Provide a high-quality, affordable service for identifying proteins, protein modifications and protein biomarkers
2. Provide consultative services and technical assistance in experimental design and implementation in protein sample preparation, separation and purification, as well as in image analysis, mass spectrometry and data mining
3. Provide education and training for basic and clinical investigators in proteomics analysis applications and capabilities
4. Provide advanced methods of sample pre-fractionation prior to mass spectrometric analysis and protein identification
5. Provide for the analysis of complex mixtures of proteins by 2D-HPLC and tandem mass spectrometry, especially for evaluating protein and chemical biomarkers for cancer and therapeutic regimens
Director: Kari Green, PhD
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