Structural fingerprinting of Asn-linked carbohydrates from specific attachment sites in glycoproteins by mass spectrometry: application to tissue plasminogen activator.
A sensitive and specific strategy has been developed for determining the sites of attachment of Asn-linked carbohydrates in glycoproteins, and defining the compositions and molecular heterogeneity of carbohydrates at each specific attachment site. In this carbohydrate 'fingerprinting' strategy, potential glycopeptides are identified by comparing the high pressure liquid chromatography (HPLC) chromatograms of proteolytic digests of a glycoprotein obtained before and after digestion with a glycosidase, usually peptide:N-glycosidase F (PNGase F). The glycopeptide-containing HPLC fractions are analyzed by fast atom bombardment mass spectrometry (FAB MS) prior to and after digestion with PNGase F to identify the former glycosylation site peptide and its sequence location (Carr and Roberts, (1986) Anal. Biochem. 157, 396-406). Carbohydrates are extracted from these fractions as the peracetates which are then permethylated and analyzed by FAB MS. The spectra exhibit molecular weight-related ions for each of the parent oligosaccharides present in the fraction which provide composition in terms of hexose, deoxyhexose, N-acetylhexosamine and sialic acid. The relative ratios of these peaks reflect the relative abundances of the various carbohydrate homologs present in the mixture. The derivatives formed are directly amenable to methylation analysis for determination of linkage. This strategy enables the structural classes of carbohydrates at specific attachment sites to be determined using only a few nmol of glycoprotein. The carbohydrate fingerprinting strategy has been applied to a number of glycoproteins including tissue plasminogen activator, the results for which are described herein.
Carr
S A
SA
Department of Physical and Structural Chemistry, Smith Kline and French Laboratories, King of Prussia, PA 19406.
Roberts
G D
GD
Jurewicz
A
A
Frederick
B
B
D000581:Amidohydrolases; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000302:isolation & purification; D002241:Carbohydrates / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D006023:Glycoproteins / Q000302:isolation & purification*; D006031:Glycosylation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010959:Tissue Plasminogen Activator / Q000302:isolation & purification*
D002241:Carbohydrates; D006023:Glycoproteins; D001216:Asparagine; D010959:Tissue Plasminogen Activator; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase