Abstract: SDS-PAGE was employed to the separation of the venomic complex of Chinese Gloydius Shedaoensis snake localized at Lüshun. The glycosylated and phosphorylated protein components were visualized by the Pro-Q Emerald 488 glycoprotein staining and Pro-Q Diamond phosphoprotein fluorescent dyes. Protein identification of the selected protein bands were performed the HPLC-nESI-MS/MS proteomic approach. Eight glycoprotein bands in the gel were identified as the homology proteins of L-amino acid oxidase, metalloproteinase, glutaminyl cyclase, salmobin, plasminogen activator, halystase, phospholipase A₂(PLA₂), nerve growth factor and truncated/degraded L-amino acid oxidase products at the N-terminus, which posses homology peptides originated from other kinds of snake venoms. The five phosphoprotein bands visualized by Pro-Q Diamond dye were identified as stejaggregin-A, PLA₂, Crisp, metalloproteinase P-Ⅲ and acutolysin e precursor homology proteins. To validate this approach, a novel PLA₂ was purified from this venom to homogeneity by the ion-exchange and gel filtration chromatography. The results from Pro-Q Diamond staining and mass spectrometry identification indicate that the purified PLA₂ sharing certain homology peptides of PLA₂s from other snake venoms. Our experimental results provide new insights for further making a research on the relationship between the post-translation modifications of snake venom proteins and their biological functions and structures.

Key words: Snake venom, Glycosylation, Phosphorylation, HPLC-nESI-MS mass spectrometry