Identification of carbohydrate structures in glycoprotein peptide maps by the use of LC/MS with selected ion extraction with special reference to tissue plasminogen activator and a glycosylation variant produced by site directed mutagenesis.
Electrospray ionization mass spectrometry utilizing a single quadrupole on line with reversed-phase HPLC (LC/MS) enables the characterization of glycoproteins in a relatively short period of time. In this approach the protein is digested with a suitable protease and the peptides are separated by reversed-phase HPLC and detected by electrospray ionization mass spectrometry. The glycopeptides are initially observed as a cluster of negatively sloping ions in a contour plot of data from the LC/MS run (m/z vs retention time) or as a characteristics series of masses at different elution times. The search for a particular glycopeptide is based on previously known carbohydrate structures and on consensus glycosylation sites. Further structural information is obtainable with glycosidase digestion and LC/MS analysis. The mass shifts following glycosidase digestion allow further confirmation of the structure. This approach identifies the site of attachment of two hybrid glycoforms to the T11 tryptic peptide in a reversed-phase tryptic map of recombinant tissue plasminogen activator (rt-PA). Use of selected ion extraction of the LC/MS data files allows one to graphically describe the elution order of closely related glycopeptides. The potential of LC/MS for the characterization of small amounts of unknown glycoproteins is shown by the study of an rt-PA mutant. A new potential site for glycosylation is created by site directed mutagenesis of wild type rt-PA with replacement of a threonine residue with asparagine at residue 103. An examination of a tryptic map shows that the mutant contains two new complex carbohydrate chains. The introduction of the new asparagine proximal to asparagine 117 changes this native high-mannose site in rt-PA to a complex-type glycosylation. This method allows rapid identification of carbohydrate containing peptides and yields useful structural information on microgram amounts of material.
Guzzetta
A W
AW
Department of Medicinal and Analytical Chemistry, Genentech Inc., South San Francisco, California 94080.
Basa
L J
LJ
Hancock
W S
WS
Keyt
B A
BA
Bennett
W F
WF
D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010959:Tissue Plasminogen Activator / Q000737:chemistry*
D006023:Glycoproteins; D010959:Tissue Plasminogen Activator