Intelligent peptide-mapping method for recombinant human tissue-type plasminogen activator by lysyl-endopeptidase digestion

A novel peptide-mapping method for recombinant human tissue-type plasminogen activator is described. It involves reduction and S-carboxyamidemethylation of the tissue-type plasminogen activator and digestion of the alkylated protein with lysyl endopeptidase, followed by liquid chromatographic analysis of the digest using a Vydac C4 column with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid. Twenty-four peptide fragments were clearly resolved, and each peptide was identified by means of a combination of amino acid analysis, automated Edman degradation, and enzymatic deglycosylation. The results indicate that the recombinant tissue-type plasminogen activator consists of 527- and 530-residue polypeptides, due to heterogeneity of N-terminal processing, and exists in one- and two-chain forms, due to limited proteolysis between Arg275 and Ile276. It contains three N-glycosylated sites at Asn117, 184, and 448, and a single O-fucosylated site at Thr61. Two variants of the recombinant tissue-type plasminogen activator were separated by hydrophobic liquid chromatography and peptide mapping of the variants demonstrated that they differed only in N-glycosylation at Asn184. Our method can thus provide information on N- and C-termini, proteolytic cleavage and glycosylation pattern of the target molecule.