Mass spectrometric characterisation of post-translational modification andgenetic variation in human tetranectin

Tetranectin, a plasminogen-binding trimeric C-type lectin-like protein prim arily involved in tissue remodeling and development, was scanned for covale nt modifications and sequence heterogeneity, using a combination of mass sp ectrometric and classical protein chemical analytical methods. Electrospray ionisation mass spectrometry showed the presence of eight components of di fferent mass and abundance in plasma tetranectin, all of higher mass than t hat calculated from the cDNA sequence. To identify and locate residues acco unting for the heterogeneity, samples of tetranectin were subjected to prot eolytic cleavage. Peptide fragments, in mixtures or in purified form, were analysed by matrix-assisted-laser-desorption-ionisation mass spectrometry a nd, where required, by Edman sequencing and compared to the cDNA sequence. Our results show that the mass heterogeneity in plasma tetranectin is due t o sequence heterogeneity at position 85 and the presence of a partially sia lylated oligosaccharide prosthetic group attached to Thr-4. Residue 85 is e ncoded in the cDNA as a Ser residue, but plasma tetranectin is a 1:1 mixtur e of Ser-85 and Gly-85 sequence variants. Mass spectrometric analysis of en zymatic and mild acid hydrolysates of an N-terminal glycopeptide showed tha t the composition and partial covalent structure of the O-linked oligosacch aride prosthetic group is less than or equal to N-acetylhexosamine less tha n or equal to [hexose, (sialic acid)(0-3)].