title: 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 Nova-Pak C18 (5 microns) column with a mobile phase that contained 0.1% aqueous trifluoroacetic acid (TFA) or 50 mM 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-beta-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 oligosaccharide is attached to Asn-117 while complex carbohydrate side-chains are attached to Asn-184 and Asn-448. The residue Asn-184 is the site of optional glycosylation that results in the formation of two rt-PA variants that contain either two or three oligosaccharides.
authors.affiliation: Genentech, Inc., South San Francisco, CA 94080.
authors.forename: R C
authors.initials: RC
authors.lastname: Chloupek
authors.forename: R J
authors.initials: RJ
authors.lastname: Harris
authors.forename: C K
authors.initials: CK
authors.lastname: Leonard
authors.forename: R G
authors.initials: RG
authors.lastname: Keck
authors.forename: B A
authors.initials: BA
authors.lastname: Keyt
authors.forename: M W
authors.initials: MW
authors.lastname: Spellman
authors.forename: A J
authors.initials: AJ
authors.lastname: Jones
authors.forename: W S
authors.initials: WS
authors.lastname: Hancock
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000032:analysis*; D010959:Tissue Plasminogen Activator / Q000032:analysis*
chemical_list: D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator