Study of the primary structure of recombinant tissue plasminogen activator by reversed-phase high-performance liquid chromatographic tryptic mapping.
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.
Chloupek
R C
RC
Genentech, Inc., South San Francisco, CA 94080.
Harris
R J
RJ
Leonard
C K
CK
Keck
R G
RG
Keyt
B A
BA
Spellman
M W
MW
Jones
A J
AJ
Hancock
W S
WS
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*
D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator