The Proteomics Shared Resource in the Biomedical Research Tower provides complete proteomic support from start to finish using the current state-of-the-art electrophoresis and imaging equipment, robotic sample handlers and mass spectrometers. We offer cell lysis of cell pellets or protein extraction from cells, protein quantitation, protein preparation (precipitation). Following protein preparation from cells or tissue, protein separation services using 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.
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. Sequence information from the MS/MS data. The resulting files are searched using Mascot Daemon by Matrix Science version 2.2.1 (Boston, MA) on a 16 node IBM blade system. Either the SwissProt database version 54.1 (283454 sequences; 104030551 residues) or NCBI database version 20071104 (5614267 sequences; 1941797005 residues) is used. Protein identifications are checked manually and proteins with a Mascot score of 50 or higher with a minimum of two unique peptides from one protein having a -b or -y ion sequence tag of five residues or better are accepted.
Core personnel provide assistance to individual investigators with the design and implementation of proteomics studies. Please contact Dr. Arpad Somogyi for a project meeting at firstname.lastname@example.org
or 614-688-0521. Our new location is 250 Biomedical Research Tower.
List of services
- 1D gel (large and mini)
- 2D gel (large and mini) Coomassie, Sypro ruby and silver staining
- Albumin removal
- Cell lysis
- Protein Precipitation
- Protein Quantitation
- DIGE labeling
- Multiplex staining
- Enzymatic digestion
- Post translational modification analysis
- Protein ID
- ITRAQ and ICAT