On-target endoglycosidase digestion matrix-assisted laser desorption/ionization mass spectrometry of glycopeptides.
The digestion of glycopeptides with endoglycosidases can be used in the process of their structural characterization, and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is often used to analyze the products of these digestions. In the currently accepted protocol for the endoglycosidase digestion of glycopeptides on the MALDI target, the target must be incubated at 37 degrees C, and an hour or more is needed for digestion. We have modified the procedure so that the process can be performed at room temperature in 5 to 15 min, and digestions are performed in the presence of a MALDI matrix. The endoglycosidases used for digestion were endoglycosidase H and peptide-N-glycosidase F. Glycopeptides from asialofetuin and endopolygalacturonase (EPG) II were used as standards because their glycan structures have been previously characterized. Glycopeptides with unknown glycan structures were also digested, including glycopeptides from pectate lyase, EPG I, and pectin methylesterase from Aspergillus niger.
Colangelo
J
J
Complex Carbohydrate Research Center, and Department of Chemistry, University of Georgia, 220 Riverbend Road, Athens, GA 30602-4712, USA.
Orlando
R
R
D000581:Amidohydrolases; D001212:Asialoglycoproteins / Q000032:analysis / Q000737:chemistry; D002265:Carboxylic Ester Hydrolases; D003373:Coumaric Acids; D060748:Fetuins; D006020:Glycopeptides / Q000032:analysis* / Q000737:chemistry; D006026:Glycoside Hydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011096:Polygalacturonase; D011133:Polysaccharide-Lyases; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013302:Streptomyces; D000509:alpha-Fetoproteins / Q000032:analysis / Q000737:chemistry
D001212:Asialoglycoproteins; D003373:Coumaric Acids; D060748:Fetuins; D006020:Glycopeptides; D000509:alpha-Fetoproteins; C017029:asialofetuin; C073734:sinapinic acid; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase; D006026:Glycoside Hydrolases; C400187:endopolygalacturonase II; D011096:Polygalacturonase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011133:Polysaccharide-Lyases; C023911:pectate lyase