Study of the primary structure of recombinant tissue plasminogen activator by reversed-phase high-performance liquid chromatographic tryptic mapping

Abstract

Two high-resolution tryptic maps have been developed for recombinant tissue plasminogen activator (rt-PA) that separate the expected 51 tryptic peptides. The trypsin digestion was performed after reduction and S-carboxymethylation of the protein. The high-performance liquid chromatographic separation of the tryptic peptides used a Nove-Pak C 18 (5 μm) column with a mobile phase that contained 0.1% aqueous trifluoroacetic acid (TFA) or 50 m M sodium phosphate (pH 2.85) and a linear gradient of acetonitrile. A TFA solvent system was also used for re-purification and for characterization of the peptides isolated from the phosphate-based separation. All of the isolated peptides had compositions consistent with the sequence proposed for rt-PA. The identities of the glycopeptides were confirmed by lectin chromatography on concanavalin A-Sepharose. The mixture of tryptic peptides was also treated with endo-β-N-acetylglucosaminidase H and peptide: N-glycosidase F to locate the position of either high mannose or complex oligosaccharides. These studies demonstrated that a high mannose is attachd to Asn-117 while complex carbohydrate side-chains are attached at Asn-184 and Asn-448. The residue Asn-184 is the site of optional glycosylation that results in the formation oft wo rt-PA variants that contain either two or three oligosaccharides.