Methods for the analysis of protein structures are being improved, applied and tested in independent and collaborative research projects in proteomics. Chromatographic systems, both on-line liquid chromatographic and gel-based are being tested. Collaborative studies have been initiated on the identification of proteins in a DNA replication complex isolated from human cells. This nuclear multi-protein complex known as the synthesome acts as a machine that synthesizes or replicates DNA. Collaborators have purified 5200-fold an intact and functional DNA replication complex from crude extracts and identified a number of the replication proteins using Western blotting. DNA polymerases alpha and gamma, DNA primase, RF-C, and the readily extractable forms of DNA ligase I and topoisomerase II were found to exclusively co-purify with the human cell-derived DNA synthesome. Gel, immunoaffinity and column chromatographic separations are being used in combination with electrospray and matrix assisted laser desorption ionization with ion trap and quadrupole/time-of-flight mass spectrometers to characterize the complex protein mixture in the synthesome. Additional separation techniques must be devised and employed because many proteins co-purify with the mammalian synthesome complex. Specific post-translational structural changes characteristic of PCNA in malignant cells requires complete primary structure characterization, whereas identification of proteins in the synthesome complex may proceed from mass spectrometric recognition of small numbers of tryptic fragments. Three additional separation projects are in progress. In the first, electrophoretic methods are being tested for the selective isolation and characterization of low molecular weight proteins not usually detected by gel based methods. In a second study, total cell homogenate proteome analyses are being performed by two-dimensional gel protein separation and in-gel tryptic digestion followed by liquid chromatography/mass spectrometry. These results are being compared with protein identifications and sequence coverage attained using a newly developed tandem liquid chromatography separation of tryptic fragments with automated mass spectrometric detection. A third study has improved methods of sample introduction and concentration for capillary zone electrophoresis. Magnetic beads that have been surface coated are washed into a column, magnetically trapped, and the sample applied. Peptides stick to the bead coating, permitting enrichment of dilute samples, consequently enhancing the applicability of capillary electrophoresis to dilute solutions.
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