Methods for the analysis of protein structures are being improved, applied and tested in independent and collaborative research projects in proteomics. An automated two dimensional liquid chromatographic system has been further improved for lower flow compatible with sub-microliter microcolumns 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. 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.
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