Comparative glycomic profiling of isotopically permethylated N-glycans by liquid chromatography/electrospray ionization mass spectrometry

Rapid Commun Mass Spectrom. 2013 Apr 30;27(8):865-77. doi: 10.1002/rcm.6512.


Rationale: Mass spectrometry based comparative glycomics is essential for disease biomarker discovery. However, developing a reliable quantification method is still a challenging task.

Methods: We here report an isotopic labeling strategy employing stable isotopic iodomethane for comparative glycomic profiling by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). N-Glycans released from model glycoproteins and blood serum samples were permethylated with iodomethane ('light') and iodomethane-d1 or -d3 ('heavy') reagents. Permethylated samples were then mixed at equal volumes prior to LC/ESI-MS analysis.

Results: Peak intensity ratios of N-glycans isotopically permethylated (Heavy/Light, H/L) were almost equal to the theoretical values. Observed differences were mainly related to the purity of 'heavy' iodomethane reagents (iodomethane-d1 or -d3). The data suggested the efficacy of this strategy to simultaneously quantify N-glycans derived from biological samples representing different cohorts. Accordingly, this strategy is effective in comparing multiple samples in a single LC/ESI-MS analysis. The potential of this strategy for defining glycomic differences in blood serum samples representing different esophageal diseases was explored.

Conclusions: LC/ESI-MS comparative glycomic profiling of isotopically permethylated N-glycans derived from biological samples and glycoproteins reliably defined glycan changes associated with biological conditions or glycoproteins expression. As a biological application, this strategy permitted the reliable quantification of glycomic changes associated with different esophageal diseases, including high grade dysplasia, Barrett's disease, and esophageal adenocarcinoma.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Barrett Esophagus / blood
  • Carbohydrate Conformation
  • Case-Control Studies
  • Chromatography, Liquid / methods*
  • Esophageal Neoplasms / blood
  • Glycomics / methods*
  • Glycoproteins / analysis
  • Glycoproteins / blood
  • Glycoproteins / chemistry
  • Humans
  • Methylation
  • Models, Chemical
  • Polysaccharides / analysis*
  • Polysaccharides / blood
  • Polysaccharides / chemistry
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*


  • Glycoproteins
  • Polysaccharides