Characterization and analysis of a novel glycoprotein from snake venom using liquid chromatography-electrospray mass spectrometry and Edman degradation

An N-linked glycosylation in a novel C-lectin protein from snake venom was observed by Edman degradation and liquid chromatography-electrospray mass spectrometry. The peptides obtained by trypsin cleavage were analyzed to confirm the amino acid sequence and Asn5 was found to be the N-glycosylation site. The result was further confirmed by N-glycosidase digestion. In addition, the protein and tryptic peptides with and without glycan chain were characterized by mass spectrometry according to the mass difference. The glycopeptide obtained from proteolytic digestion was analyzed and the glycoforms were identified as high-mannose type by tandem MS coupled with a-mannosidase digestion. An oxidized Met residue was detected and located in the protein by mass spectrometry. Glycosylation is a post-translational processing frequently observed in proteins. The diversity of monosaccharides and the variety of linkages give rise to the complexity of glycoproteins. Glycosylation plays an important role in the biological properties of many proteins, but brings about difficulties in their characterization. Reverse-phase high-performance liquid chromatography- electrospray ionization mass spectrometry (LC-ESI-MS) has been shown to be a rapid and sensitive means for characterizing the primary structure of proteins and their post-translational modifications including glycosylation (1-7). To determine the site of glycosylation and site-specific microheterogeneity of glycoprotein, endoproteases are usually employed to obtain the glycopeptides which are separated by HPLC and identified by electrospray mass spectrometry. LC-ESI-MS coupled with protease and glycanase (such as N-glycosidase F) digestion can identify the sites of glycosylation, detect whether a glycan is N-linked or O-linked, and even differentiate high-mannose, complex and hybrid forms of N-linked glycan. C-type lectins (8) are sometimes glycoproteins themselves (9,10). A novel C-type lectin glycoprotein (11) was isolated from the venom of Trimeresurus stejnegeri and designated as TSL. The protein sequence, deduced from cDNA, is KYKPG- CHLASFHRLAESLDIAEYISDYHKRQAEVWIGLLDRKKDF- SWEWTDRSCTDYLNWDKNQPDHYKDKEFCVELVSLTG- YHRWNDQVCESKNSFLCQCKF. The protein is composed of 135 amino acids, with a calculated mass of 16 207.3 Da. The N-terminal 30-amino acid residues except residue 5, a potential N-glycosylation site, were sequenced by Edman degradation. In addition, the molecular mass measured by mass spectrometry was not consistent with the calculated value, possibly as a result of glycosylation. In this paper, by mass spectrometry and Edman degradation, coupled with protease and glycosidase digestion, we report the post-translational modifications of TSL, including the site of N-glycosylation and the structure of glycan chains attached to the protein.