Comparative Glycomic Mapping through Quantitative Permethylation and Stable-Isotope Labeling

Abstract

Comparative glycan quantification has thus far been a challenging task due to the lack of sensitive and reproducible analytical techniques. We introduce here a combination of quantitative permethylation and isotope labeling of glycans as an approach (C-GlycoMAP) allowing precise comparison between different samples in a single MALDI-MS analysis. Samples are either methylated or deuteriomethylated prior to their mixing and mass spectrometric acquisitions. Comparative analyses are based on the ratio of the two isotopically distinct forms of the same glycan structure, thus allowing a direct absolute evaluation of the intensities of the two forms originating from two different biological samples (e.g., control and diseased). The direct comparison between the two forms eliminates a MALDI-MS low m/z bias commonly associated with this technique. These comparative analyses are highly reliable when the intensity ratios of the two forms lie between 0.125 and 6, an overall reproducibility better than 30% (RSD). The value of C-GlycoMAP is demonstrated here for N-glycans derived from human blood serum collected from a healthy individual and a breast cancer patient as well as for O-glycans derived from normal and cancer cell extracts.