Metabolic Isotope Labeling of Polysaccharides with Isotopic Glucose for Quantitative Glycomics in Cell Culture

Abstract

The attachment of sugars to the side chains of proteins via glycosylation is a common post-translational modification that plays significant roles in a wide range of biological processes in living organism. 1,2 The majority of extracellular matrix and the membranous proteins are highly glycosylated by N-linked glycans as well as other types of glycans. 3 A change in glycome profiles and the expression levels of proteins responsible for glycan complexity has been reported in a variety of disease states and during the course of developmental processes. [4][5][6] In order to understand the biological relevance of glycans, many mass spectrometry (MS)-based methods have been developed for the relative quantitation of glycan. The simplest strategy is a label-free method in which the quantification is relied on the prevalence of each glycan signal in order to calculate the proportion of an individual glycan to the total glycans in the sample. 7 Several in vitro chemicalbased isotopic labeling methods have also been developed to introduce signature tags at the reducing end of N-linked glycans. 8,9 Another version of relative isotopic labeling of glycans utilizes heavy vs. light iodomethane ( 13 CH 3 vs. 12 CDH 2 ) in a standard permethylation workflow for the incorporation of isobaric structures. 10-12 Also, it is possible to incorporate 18 O-atoms at the reducing end of N-glycans during deglycosylation of enzyme glycosidase in order to achieve the purpose of quantification. 13,14 In contrast to the aforementioned in vivo labeling methods, a metabolic labeling method, isotopic detection of aminosugars with glutamine (IDAWG), has been reported by Wells and coworkers for comparative glycomic studies using the murine embryonic stem cells. 15,16 In this strategy, amide-15 N glutamine is used as sole donor source of nitrogen for aminosugars in the production of nucleotide sugars through the hexosamine biosynthetic pathway.Herein, we describe a novel in vivo labeling method using either light or heavy glucose (1-13 C 1 ) to tag glycans from rice (Oryza sativa) culture. As shown in Figure 1(a), isotopic glucose enters the glucose metabolic pathway, which in turn produces nucleotide sugar precursors that are subsequently used for all glycan biosynthesis. We named this method Metabolic Isotope Labeling of Polysaccharides with Isotopic Glucose (MILPIG) and provide evidence for the quantitative power of MILPIG on N-linked glycans after 2 weeks on labeling (Figure 1(b)). In addition, we foresee that other glycoconjugates such as O-linked glycans, glycolipids, and extracellular matrix polysaccharides should be susceptible to MILPIG labeling. We validated the application of this approach by MSbased analysis of N-linked glycans released from proteins of rice cultures grown in both light and heavy glucose. Figure 2 shows the full mass spectra of N-linked glycans prepared by mixing equal amounts of normal and heavy labeled samples. The isotopic pairs of N-linked glycans on the full MS demonstrate a robust incorporation of 1-13 C 1 glucos...

Analysis of Carbohydrates and Glycoconjugates by Matrix‐assisted Laser Desorption/Ionization Mass Spectrometry: An Update for 2015–2016

“…The products were appropriate for quantitative glycomic studies in cell culture. The method was named Metabolic Isotope Labelling of Polysaccharides with Isotopic Glucose (MILPIG) (Seo et al, 2016).…”

Isotope Labeling of N‐linked Glycans by Transglycosylation of Endo‐M and Isotopic Glucose for Quantitative Glycomics

“…Glycan reducing‐end 18 O‐labeling (GREOL) strategy using 18 O‐water was developed as an enzymatic in vitro labeling approach 22,23 . In addition, isotopic detection of aminosugars with glutamine (IDAWG) and metabolic isotope labeling of polysaccharides with isotopic glucose (MILPIG) have been developed as metabolic isotope labeling strategies for glycans, with glutamine or glucose, respectively, as the source of the isotope label 24,25 …”