Mass spectrometry study of ecto‐5′‐nucleotidase from bull seminal plasma

The structure of ecto‐5′‐nucleotidase from bull seminal plasma, containing a glycosyl‐phosphatidylinositol anchor, was studied using mass spectrometry. MALDI‐MS analysis of intact protein indicated a mass of 65 568.2 Da for the monomeric form, and it also showed a heterogeneous population of glycoforms with the glycosidic moiety accounting for ≈ 6000 Da. MALDI‐MS analysis showed that Asn53, Asn311, Asn333 and Asn403 were four sites of N ‐glycosylation. GC‐MS analysis provided information on the glycosidic structures linked to the four asparagines. Asn53, Asn311 and Asn333 were linked to high‐mannose saccharide chains, whereas the glycan chains linked to Asn403 contained a heterogeneous mixture of oligosaccharides, the high‐mannose type structure being the most abundant and hybrid or complex type glycans being minor components. By combining enzymatic and/or chemical hydrolysis with GC‐MS analysis, detailed characterization of the glycosyl‐phpsphatidylinositol anchor was obtained. MALDI spectral analysis indicated that the glycosyl‐phosphatidylinositol core contained EtN(P)Man 3 GlcNH 2 ‐myo‐inositol(P)‐glycerol, principally modified by stearoyl and palmitoyl residues or by stearoyl and myristoyl residues to a minor extent. Moreover, 1‐palmitoylglycerol and 1‐stearoylglycerol outweighed 2‐palmitoylglycerol and 2‐stearoylglycerol. The combination of chemical and enzymatic digestions of the protein with the mass spectral analysis yielded a complete pattern of S–S bridges. The protein does not contain free thiols and its eight cysteines are linked by intramolecular disulfide bonds, the pairs being: Cys51–Cys57, Cys353–Cys358, Cys365–Cys387 and Cys476–Cys479. This work resolves details of the structure of ecto‐5′‐nucleotidase, with particular regard to the localization and composition of the glycidic moiety, number and localization of the disulfide bridges and characterization of the glycosyl‐phosphatidylinositol anchor.