The bioactive protein components from snake venom complexes have been utilized for studies of enzymology, structural biology, and pharmacology. The Gloydius shedaoensis snake (GSS) is the only snake species found exclusively at the Chinese Shedao (snake) Island in Dalian. To investigate the protein components of Chinese GSS venom (GSSV), we initialized a proteomic assay for GSSV by the combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis with high-performance liquid chromatography (HPLC)-nanoelectrospray ionization tandem mass spectrometry (nESI-MS/MS). Thirty gel bands visualized by Coomassie blue staining were excised and digested by trypsin. The tryptic-digested peptides were separated by HPLC and subsequently sequenced by nESI-MS/MS. Twenty-four types of proteins were identified by searching the mass spectrometry data against NCBInr database through TurboSequest Bioworks. The most abundant proteins are phospholipase A(2), metalloproteinase, L-amino acid oxidase (LAAO), serine protease/thrombin-like enzyme. Except for 20 types of known snake venom proteins, the homolog peptides of hypothetical protein PFLC2230, LOC495267 protein, DEAD/DEAH box helicase-like, and pancreatic trypsin 1 from other organisms are matched for GSSV protein components. Mass spectrometric data also indicated that (i) dimerization happens to PLA(2)s as monomer and dimer of PLA2s coexist in GSSV and (ii) truncation or hydrolysis might happen to LAAOs as three molecular-weight-ranged LAAO species are present in GSSV. The results provide an "anatomical" view of the protein composition and important information for protein characteristics of GSSV. Anat Rec, 294:273-282, 2011. (C) 2010 Wiley-Liss, Inc.