Application Of Biological Mass Spectrometry In Scorpion Peptide Toxin Identification And Method Development For Post-translational Modification Analysis

Proteomics holds tremendously potential in the post-genomic era. Mass spectrometry-based proteomics not only important for large-scale protein identifications, it is also especially powerful in analysis of posttranslational modifications. Proteomics offers an important tool in systematically understanding of biological process. The three central components in proteomics techniques include: sample preparation and chromatograph separation, mass spectrometry and bioinformatics. Recent advances in proteomics technology, including new enrichment methods, high-accuracy mass spectrometry and more powerful bioinformatics, will promote studies in system biology.In this paper, we described studies on the two areas:1) Utilizing proteomics to identify proteins from venoms of Asian scorpion Buthus martensi Karsch and Asian forest scorpion. In this study we combined proteomics and transcriptomics technologies. Not only we verified the existence of many venom proteins, we also analyzed functional classifications of these proteins.2) We explored methods in identifications of protein post-translational modifications, such as the enrichment and determination of phosphorylated peptides, MIDAS (MRM-initiated detection and sequencing)-based method for the identification of protein nitrosylation. Using as medicine over a thousand years in our country, scorpion venoms are composed of many toxin peptides with potential medicinal functions. Through modern science and technology, the biological mechanisms for some venom peptides were elucidated, and some toxins have already entered into clinical trials. The advancement in proteomics techniques, such as better chromatograph separation and mass spectrometry detection, is providing better tools for scorpion venom research. We carried out a comprehensive analysis of venom peptides for the Asian scorpion Buthus martensi Karsch and the Asian forest scorpion by combining chromatograph separation with mass spectrometry technology. We used multidimensional chromatograph to fractionate scorpion venom components. Purified single peptide was sequenced by Edman degrading, and molecular weights for other fractions were determined by MALDI-TOF. Two-dimensional electrophoresis was also used to exam and compare in the distributions of larger scorpion proteins. Through construction of a high quality cDNA library of Asian forest scorpion venom using transcriptome data, we applied "shotgun" proteomics techniques for "bottom-up" analysis of venom peptides. Our work offers a new approach for venom research, and proves that the online multidimensional chromatograph mass spectrometry techniques are effective in low abundant toxin analysis.Post-translational modifications are involved in many regulatory functions such as signal transduction, cell differentiation and metabolism. With broad interest, protein phosphorylation is one of the most important post-translational modifications, and it reversibly regulates almost all biological processes. We synthesized several different materials for the enrichment of phosphopeptides, and compared their selectivities. Through optimization of synthesis process and elution condition, we try to improve phosphopeptide detection. We also carried out a related work using MIDAS method to detect protein.