Proteomics

Proteomics Shared Resource Facility

Our facility provides comprehensive proteomic analysis services using state-of-the-art equipment, including:

• Advanced electrophoresis systems
• Modern imaging equipment
• Robotic sample handlers
• 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.

High-Precision Protein Analysis Using Advanced Mass Spectrometry

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.

List of Services

• 1D gel (large and mini)
• 2D gel (large and mini) 
• Albumin removal (MARS Column)
• Cell lysis
• Coomassie, Sypro ruby and silver staining
• Differential Gel Electrophoresis (DIGE) labeling
• Enzymatic digestion
• IP pulldowns
• ITRAQ and ICAT
• LC-MALDI
• LC-MS/MS
• MALDI imaging
• MudPIT
• Multiplex staining (phosphorlyation and glycosylation analyses)
• N-Terminal Sequencing
• OFFGEL fractionation
• Post-translational modification analysis
• Protein ID
• Protein precipitation
• Protein quantitation
• Quantitative proteomics using label free quantitation or iTRAQ
• Western Blot