Protein Analysis And Identification Using ElectroSpray Ionization Tandem Mass Spectrometry

The enzymatic digest of bovine serum albumin(BSA),used as a model protein, was directly analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC)/electrospray tandem mass spectrometry (ESI-MS/MS). The resulting MS and MS/MS data, after being processed with analytical software under different parameters, were used for the protein identification through database search with three different methods. All the three methods could identify the protein correctly. Peptide mass fingerprint(PMF) was the simplest and most direct, with its search results being easily influenced by parameter changes during the data process and database search. Using raw MS/MS data could obtain correct identification result in wider change range of database search parameters. The Sequence Query (SQ) utilizing the de novo sequencing results could make steady and definite identification based on the data of a few of peptides, which was hardly influenced by the main database search parameter changes. The effects of enzymatic digestion, data process and database search parameters on the identification of proteins were discussed in detail, which provided references for the proteomics researches.Jingzhaotoxin-VIII and Huwentoxin-VI are peptideneurotoxins purified from venom of spiders Selenocosmia huwena and Chilobrachys jingzhao, respectively. The most of amino acid sequences were determined by Edman degradation, with a few of sequences uncertained due to the ambiguous signals. In order to identify the uncertain parts, the two toxins were digested by trypsin and then analyzed by using ESI-Q-TOF mass spectrometry. By interpreting the resulting MS and MS/MS spectra related to the uncertain sequences with analytical software and by hand, the amino acid sequences of uncertain parts were clearly identified. The research shows that, compared with contraditional methods, the de novo sequencing using mass spectrometry has particularadvantages in structural analysis and identification of proteins and peptides, and can be used as a good complement for Edman degradation. Membrane proteins perform some of the most important functions in the cell. The proteomic definition of plasma membrane proteins is an important initial step in searching for novel tumor marker proteins expressed during the different stages of cancer progression. Nasopharyngeal carcinoma (NPC) is a commonly occurring tumor in southern China and south east Asia. We purified the crude plasma membrane from a human nasopharyngeal carcinoma cell line. After two dimensional gel electrophoresis separation, silver staining, ESI-Q-TOF analyses, tryptic peptide MS/MS were searched for matches in the SWISS-PROT, NCBI and Mascot databases. Over 120 spots were identified using this approach (including 92 membrane proteins). These proteins include those involved in regulation of gene expression, affected the signal pathway, altered the cell metabolism, inhibit or accelerate cell proliferation, induce the cell apoptosis and intervened the tumor invasion. This study of the NPC proteome, coupled with similar proteome analyses of the whole cell, the normal nasopharyngeal tissues, and other nasopharyngeal carcinoma cell lines, not only defines the proteome of the NPC membrane for the first time, but also represents the first step towards the establishment of plasma membrane protein databases, which are valuable resources in studies on the differential protein expressions of human nasopharyngeal carcinoma.