Simplifying the exoglycosidase digestion/MALDI-MS procedures for sequencing N-linked carbohydrate side chains.
Exoglycosidase digestion coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an effective technique for sequencing the N-linked carbohydrate side chains of a glycoprotein. However, the buffers currently used in the enzymatic procedures are detrimental to MALDI-MS, and thus desalting is required before the digestion products can be analyzed. We demonstrate that a 25 mM ammonium acetate solution adjusted to the proper pH can replace the normal exoglycosidase digestion buffers. The use of these ammonium acetate solutions permits direct MALDI-MS analysis of the digestion mixture without desalting. More importantly, we show that many of the commonly used exoglycosidases retain both their activity and their specificity under these conditions.
Yang
Y
Y
Complex Carbohydrate Research Center, University of Georgia, Athens 30602-4712, USA.
Orlando
R
R
D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis*; D006023:Glycoproteins / Q000032:analysis*; D006026:Glycoside Hydrolases / Q000737:chemistry*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002241:Carbohydrates; D006023:Glycoproteins; D006026:Glycoside Hydrolases