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MSRC Proteomics Lab

MSRC Proteomics Laboratory Services

Simple Protein Identification

This service provides identification of proteins from 2D gel spots, from 1D gel bands of simple mixtures and from nominally pure proteins.  Core personnel do the gel-excision, in-gel or in-solution digestion with trypsin or other proteases, elute the peptides from the gel, perform a additional cleanup as necessary, and analyze the peptide mixture by MS/MS using either the LTQ or MALDI-TOF/TOF systems and associated bioinformatics tools to identify the protein(s).  Protein identification is based on surrogate peptides analyzed by MS/MS, and these experiments can be performed in a qualitative/quantitative fashion using either stable isotope labeling or spectral counting methods.  Please contact Proteomics Laboratory personnel for more details.

Multidimensional LC-MS/MS (MudPIT)

Multidimensional Protein Identification Technology (MudPIT) couples extended HPLC separations with sensitive MS/MS analysis of resolved peptides using either the LTQ or high-resolution Orbitrap MS systems.  It is the most powerful technique available to proteome mining of complex mixtures, and often provides enhanced sensitivity and dynamic range for more complex protein mixtures than the single-stage reverse phase HPLC separation used for our standard protein identification service listed above.  MudPIT analysis incorporates an additional peptide separation by strong cation exchange upstream of reverse phase HPLC, and these two separations are coupled directly in-line for automated analysis over a 24-hour period.  Protein identification is based on surrogate peptides analyzed by MS/MS, and these experiments can be performed in a qualitative/quantitative fashion using either stable isotope labeling or spectral counting methods.  Please contact Proteomics Laboratory personnel for more details.

Specialized/Targeted Analyses using Mass Spectrometry

The Proteomics Laboratory routinely embarks on specialized research problems that go beyond the standard platforms listed above, but that utilize these technologies. Such experiments may include mapping sites of post-translational modification (e.g., phosphorylation, ubiquination, cross-linking) and mapping stable domains of proteins for subsequent structural analysis (NMR, X-ray crystallography). Please contact Proteomics Laboratory personnel for more details.

2D Difference Gel Electrophoresis (2D-DIGE)

This technique is used for relative quantification of expression changes from simple, complex and unfractionated extracts. Unlike the peptide-based technologies listed above, the DIGE platform offers relative quantification on intact proteins (included charged-isoforms, splice variants and proteolytic products).  It is most often used to detect global changes in patterns of protein expression in response to a single or multiple experimental conditions, and can accommodate the necessary individual replicate samples to provide a statistically-powered experiment.  It uses three spectrally-resolvable cyanine dyes (Cy2, Cy3 and Cy5), where each DIGE gel contains a tripartite mixture of two individual samples pre-labeled with Cy3 or Cy5 and an aliquot of a Cy2-labeled mixed-sample internal standard.  As many gels are run as are needed to accommodate all of the individual samples, enabling single- or multi-variable analyses with repetitive samples to be performed in one concerted experiment.  Analysis of DIGE data is performed using the DeCyder suite of software tools. The DIGE gels are post-stained with SyproRuby to ensure accurate excision of proteins of interest for subsequent identification using mass spectrometry and database interrogation.  Please contact Proteomics Laboratory personnel for more details.

This page was last updated October 1, 2009 and is maintained by