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.

Tandem Mass Spectrometry of Tagged and Permethylated Polysaccharides

“…The sample was stored at -20 °C prior to analysis. An example of this labeling approach is demonstrated using a comparative glycoform mapping method (C-GlycoMAP), developed based on differential stable isotope labeling (Kang et al, 2007). The differentially isotope labeled samples are combined prior to MALDI-TOF MS analysis to minimize sample-to-sample variation in peak abundances and maximize the ability to perform the comparison of samples.…”

Untitled

“…Isotope labeling oligosaccharides in the mass spectrometric analyses is also quite useful for the identiˆcation of glycosylation sites (69)(70)(71) and quantitative analysis of oligosaccharide mixtures (72,73). It is, however, not feasible to determine the oligosaccharide structure unambiguously solely on the MS data because there exist a variety of isomeric structures with diŠerent composition, glycosidic linkage and branching.…”

Mass spectrometric analysis of the glycosphingolipid‐derived glycans from miniature pig endothelial cells and islets: identification of NeuGc epitope in pig islets

“…[28] However, MSbased quantification by multistep fragmentation is unsuitable for quantitative analysis of complex glycan mixtures. Permethylation can be used to minimize the different ionization efficiencies of complex oligosaccharides and for relative quantification by MS. [29] However, it still may not be enough to avoid the complicated mixture of metal adducts, such as [M+K] + and [M+Na] + . Herein, the GSL-derived glycans obtained after the small-scale delipidation and desalting steps were partially permethylated to identify both neutral and acidic glycans using MALDI-TOF MS and ESI-MS/MS.…”