Characterization of cellobiohydrolase I N-glycans and differentiation of their phosphorylated isomers by capillary electrophoresis-Q-Trap mass spectrometry

Anal Chem. 2004 Oct 1;76(19):5878-86. doi: 10.1021/ac0493976.

Abstract

A capillary electrophoresis-mass spectrometric (CE-MS) method is described for the simultaneous analysis of uncharged and charged glycans. The glycans were labeled with the negatively charged tag 8-aminopyrene-1,3,6-trisulfonate by reductive amination and separated in an ammonium acetate buffer. A Q-Trap instrument was used for mass spectrometric detection. The CE-MS method was first optimized using maltooligosaccharides and ribonuclease B N-glycans and then applied to the characterization of enzymatically released N-glycans from the glycoprotein cellobiohydrolase I. The method, as developed, allowed differentiation of phosphorylated isomers and MS/MS provided useful structural information. Further structural evidence was obtained by studying the methylated glycans in off-line ESI-MS/MS experiments and by using a combination of chemical and enzymatic sequencing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carbohydrate Conformation
  • Cellulose 1,4-beta-Cellobiosidase / analysis*
  • Cellulose 1,4-beta-Cellobiosidase / chemistry*
  • Cellulose 1,4-beta-Cellobiosidase / metabolism
  • Electrophoresis, Capillary / methods*
  • Ions / chemistry
  • Isoenzymes / analysis
  • Isoenzymes / chemistry
  • Isoenzymes / metabolism
  • Mass Spectrometry / methods*
  • Phosphorylation
  • Polysaccharides / analysis*
  • Polysaccharides / chemistry*
  • Polysaccharides / metabolism

Substances

  • Ions
  • Isoenzymes
  • Polysaccharides
  • Cellulose 1,4-beta-Cellobiosidase