Determination of the complete covalent structure of the major glycoform ofDQH sperm surface protein, a novel trypsin-resistant boar seminal plasma O-glycoprotein related to pB1 protein

The complete covalent structure of a novel boar DQH sperm surface protein r esistant to many classical procedures of enzymatic fragmentation was determ ined. The relative molecular mass of the major form of this protein determi ned by ESI-MS and MALDI-MS was 13,065.2 +/- 1.0 and 13,065.1, respectively. However, additional peaks differing by 162 Da (i.e., minus hexose), 365 Da (i.e., minus hexose and N-acetylhexosamine), 146 Da (i.e., plus deoxyhexos e), and 291 Da (i.e., plus sialic acid) indicated the heterogeneity due to differences in glycosylation. The complete covalent structure of the protei n was determined using automated Edman degradation, MALDI-MS, and post-sour ce decay (PSD) MALDI-MS, and shown to consist of N-terminal O-glycosylated peptide followed by two fibronectin type II repeats. The carbohydrates are O-glycosidically linked to threonine 10, as confirmed by PSD MALDI-MS of th e isolated N-terminal glycopeptide. Eight cysteine residues of the protein form four disulfide bridges, the positions of which were assigned from MALD I-MS and Edman degradation data. We conclude that mass spectral techniques provide an indispensable tool for the detailed analysis of the covalent str ucture of proteins, especially those that are refractory to standard approa ches of protein chemistry.