Methods In Mass Spectrometry

National Institute Of Mental Health

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Abstract

Mass spectrometric methods for the analysis of protein structures are being improved, applied and tested in independent and collaborative research projects in proteomics. Projects in separation methods, derivatization chemistry, and data processing are in progress. An automated two dimensional liquid chromatographic system has been developed over the past two years and is now employed routinely for the analyses of complex mixtures of peptides using nano-electrospray ionization mass spectrometry. The system fractionates using strong cation exchange chromatography, followed by gradient elution on a single reverse phase column. The system has been tested with soluble proteins from yeast and is capable of identifying hundreds of proteins in the resulting complex extract. Extensive evaluation of the microspray 2D-LC system has been performed to define optimum system capacity and fractionation conditions. LC-MS/MS peptide analyses routinely generate large data sets leading to long lists of identified peptides and proteins using automated interpretation programs. The resulting files contain relevant experimental information, but subsequent sorting, collation, and comparison of these results pose significant tasks when analyzing multiple files, especially when summary reports are desired. We have developed software collaboratively that facilitates multiple file interpretation and comparison. A perl program (DBParser) retrieves output from proprietary searching program flat files and stores data in a new database. Protein summaries are based upon the principle of parsimony, identifying the minimal set of proteins that can explain all of the observed peptides. The program generates user-friendly html output reports of the sorted and compared peptide/protein lists which can be used for subsequent analysis. A CGI-based graphical user interface allows execution of DBParser over a network using a web browser to facilitate its use by multiple laboratories. DBParser has been refined to include quantification. A new technique to determine protein stoichiometry in macro-protein complexes is being developed and tested. Protein complexes isolated from mitochondria or sub-cellular organelles are being explored to determine the best methods of isolation and analysis. Procedures are being tested to find fluorescent peptide derivatives suitable for quantification and comparison of complex mixtures.