Speaker: Salvatore Sechi, National Institute on Aging, Baltimore, MD 21224

Topic: Modification of cysteine residues - A tool in proteomics

Place: Building 426, Conference Room, NCI-Frederick, Frederick, MD

Time: Tuesday, July 10, 2001, at 2:00 PM

Abstract: Gel electrophoresis is often used for the primary analysis and purification of proteins. Peptide mapping by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a widely used technique for the rapid identification of unknown proteins isolated by gel electrophoresis and more generally for protein characterization. The identification is usually obtained by digesting the protein with an enzyme and matching the masses of the proteolytic peptides with those of each protein in a sequence database. Sometimes more than one candidate for the identity of the protein can be found, whereupon additional information obtained by MS-MS is generally used to constrain the search. However, this requires a more complex and time-consuming analysis of the sample. An alternative method to constrain the database search to fewer candidates is to input more information about the peptides derived from the protein digest. In this presentation I describe a methodology that significantly enhances peptide mapping of proteins isolated by gel electrophoresis. In particular I compare the use of several alkylating reagents and show that using a mixture of acrylamide and deuterium labeled acrylamide the cysteine content for each peptide in the map can be determined. The presented procedure has the added benefit of reducing the sample handling and of increasing the alkylation efficiency.

Several methodologies that use isotope labeling of proteins for quantitative proteomic studies have been recently introduced (e.g. using ICAT reagents or growing cells in isotopically enriched nutrients). In this presentation I will briefly discuss pros and cons of some of these methodologies and present a simple alternative that allows the comparison of two complex mixtures without any additional step to the standard procedure for peptide mapping.