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

Authors


Correspondence to: Y. Mechref, Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA.

E-mail: yehia.mechref@ttu.edu

Abstract

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. Copyright © 2013 John Wiley & Sons, Ltd.

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