Relative Quantitation Of HeLa Cell N-Glycans Using Deuterium Oxide Labeling For Global Omics Relative Quantification(DOLGOReQ)

Abstract

Introduction Glycosylation is a well-known PTM, which have been implicated in protein folding, immune recognition, and intercellular interactions. Relative quantification of glycans can be performed to assess the glycosylation changes under varying conditions. Typically, metabolic labeling using stable isotope labeled monosaccharides or chemical labeling targeting glycan moieties is exploited for relative quantification of glycans. However, metabolic labeling based on isotope labeled precursors can be only applied to study specific biological pathways and chemical labeling suffers from side reactions or incomplete derivatization. In this study, partial metabolic deuterium oxide labeling for relative glycan quantification were evaluated in terms of quantification accuracy and precision. Methods For deuterium oxide labeling for global omics relative quantification (DOLGOReQ), glycans were prepared from HeLa cells cultured for 48 h on normal and D2O enriched media. To evaluate the performance of relative quantification of glycans, unlabeled and labeled HeLa cells were mixed in various ratios by their cell numbers. Equimolar mixtures were prepared in three replicates for the assessment of reproducibility. Plasma membrane proteins was isolated, and N-glycans were enzymatically released by N-glycosidase F. LC-MS/MS was performed for unlabeled, mixed, and D-labeled glycans for measuring the isotopic distribution and identification using their accurate m/z and CID fragmentation patterns. Glycans with their own unique retention times and m/z were measured for their fold changes and the degree of D-labeling using DOLGOReQ. Preliminary Data or Plenary Speakers Abstract In order to assess the compatibility of DOLGOReQ platform for glycan quantification, deuterium labeling efficiency, and reproducibility and accuracy for quantification were measured. Thirty-six N-Glycans were identified from their CID patterns by searching for m/z in various combinations of four monomers: hexose, hexose with N-acetyl group, fucose, and sialic acid. When glycans with the same m/z were found at different retention times, each was -treated as a different glycan. For deuterium labeling efficiency, 4% D2O media showed quantifiable isotopic distribution shifts without significant increase in the number of mass isotopomers. The average H-D distance, which is a functional index of the degree of D-lableing, of quantified glycan was 0.244 (0.197- 0.295). No significant relationship was found between H-D distance and monomer compositions constituting glycans. It was also confirmed that DOLGOReQ could be used robustly for variable glycans with constant labeling efficiency. DOLGOReQ for three biological replicates of equimolar mixture between unlabeled and D-labeled glycans revealed quantification reproducibility and accuracy comparable with relative quantification of lipids by DOLGOReQ. The dynamic range of DOLGOReQ for glycan was determined by comparing the quantitative results of the glycan mixtures from 20:1 to 1:20. Finally, DOLGOReQ for glycan was applied to real biological samples for verification of the feasibility. Novel Aspect Application of partial metabolic deuterium oxide labeling to relative quantification of glycans on a global scale