Methods Of Ionization In Mass Spectroscopy

National Institute Of Mental Health

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Abstract

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 designed and implemented for the analyses of complex mixtures of peptides using electrospray ionization mass spectrometry. The system fractionates using strong cation exchange into six traps, 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. Collaborative studies have been continued on the identification of nuclear proteins in a DNA multi-protein replication complex isolated from human cells. Collaborators isolate an intact and functional DNA replication complex from cell extracts. We have confirmed the presence of 11 known replication proteins but further separation steps are required to reduce protein background. Two dimensional liquid chromatography with various proteolytic enzymes have been used. Many proteins co-purify with the mammalian synthesome complex, and >200 have been identified. One component of the synthesome complex is PCNA. There appear to be specific post-translational structural changes in PCNA characteristic of malignant cells. Methods for the isolation of PCNA are being pursued, including micro scale solution isoelectric focusing.