Convenient and Precise Strategy for Mapping N-Glycosylation Sites Using Microwave-Assisted Acid Hydrolysis and Characteristic Ions Recognition

Anal Chem. 2015 Aug 4;87(15):7833-9. doi: 10.1021/acs.analchem.5b02177. Epub 2015 Jul 23.


N-glycosylation is one of the most prevalence protein post-translational modifications (PTM) which is involved in several biological processes. Alternation of N-glycosylation is associated with cellular malfunction and development of disease. Thus, investigation of protein N-glycosylation is crucial for diagnosis and treatment of disease. Currently, deglycosylation with peptide N-glycosidase F is the most commonly used technique in N-glycosylation analysis. Additionally, a common error in N-glycosylation site identification, resulting from protein chemical deamidation, has largely been ignored. In this study, we developed a convenient and precise approach for mapping N-glycosylation sites utilizing with optimized TFA hydrolysis, ZIC-HILIC enrichment, and characteristic ions of N-acetylglucosamine (GlcNAc) from higher-energy collisional dissociation (HCD) fragmentation. Using this method, we identified a total of 257 N-glycosylation sites and 144 N-glycoproteins from healthy human serum. Compared to deglycosylation with endoglycosidase, this strategy is more convenient and efficient for large scale N-glycosylation sites identification and provides an important alternative approach for the study of N-glycoprotein function.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Biomarkers / analysis
  • Glycoproteins / analysis
  • Glycoproteins / blood*
  • Glycoproteins / chemistry
  • Glycosylation
  • Humans
  • Hydrolysis
  • Ions / analysis*
  • Mass Spectrometry
  • Microwaves*
  • Molecular Structure


  • Biomarkers
  • Glycoproteins
  • Ions