| Proteome has been defined as the PROTEin complement expressed by a genOME or tissue. Proteomics is the field that involves the identification, characterization, and quantification of proteins in tissues or whole cells. In an ideal situation, protein characterization would likely include sequence analysis and cellular localization, plus identification of post-translational modifications, splice variants, and binding partners. In recent years, mass spectrometric methods have become an essential tool for proteomics research.; In the first project, proteins involved in yeast 66S ribosome assembly intermediates were identified from a complex mixture using nano-flow HPLC (nHPLC) microelectrospray ionization (μESI) mass spectrometry. 66S particles were affinity purified and digested with trypsin and the resulting peptide mixture was analyzed by nHPLC/μESI/MS/MS. A total of 115 proteins were identified. The role of some of these proteins was examined by determining their subcellular location and by assaying the effects of depleting these proteins on production of 60S subunits.; The second project involved identification of cargoes of nuclear import factors (Karyopherins, or Kaps) and the Kaps that import histories. The binding partners of 5 Kaps were affinity purified and digested with trypsin. The resulting peptide mixture was analyzed by mass spectrometry. The Kaps that import histories H2A, H2B, H3 and H4 were purified in the same manner as above, analyzed by mass spectrometry and confirmed by biological techniques. The post-translational modifications of H3 and H4 in the cytosol were characterized by mass spectrometry and the effects of these modifications on the nuclear localization of H3 and H4 were studied.; The phosphoproteome analysis of Arabidopsis thaliana was reported in the third project. In Arabidopsis thaliana , there is evidence that phosphorylation plays an important role in signal transduction cascades. However, signal pathways are not known and not much work has been done at the protein level to identify the substrates of these protein kinases. In this work, total protein extract of Arabidopsis thaliana was digested with trypsin. The phosphopeptides were enriched by immobilized metal affinity chromatography (IMAC) and analyzed by mass spectrometry. About 740 phosphopeptides were detected. Sequences for 78 peptides have been confirmed to date. |
