8-plex LC–MS/MS Analysis of Permethylated N-Glycans Achieved by Using Stable Isotopic Iodomethane

Abstract

Glycosylation is an important post-translational modification of proteins. Many diseases, such as cancer have proved to be related to aberrant glycosylation. High throughput quantitative methods have gained attention recently in the study of glycomics. With the development of high-resolution mass spectrometry, the sensitivity of detection in glycomics has largely improved; however, most of the commonly used MS-based techniques are focused on relative quantitative analysis, which can hardly provide direct comparative glycomic quantitation results. In this study, we developed a novel multiplex glycomic analysis method on an LC-ESI-MS platform. Reduced glycans were stable isotopic labeled during the permethylation procedure, with the use of iodomethane reagents CH 2 DI, CHD 2 I, CD 3 I, 13 CH 3 I, 13 CH 2 DI, 13 CHD 2 I, 13 CD 3 I, and CH 3 I. Up to 8-plex glycomic profiling was possible in a single analysis by LC-MS, and a 100k mass resolution was sufficient to *

Recent Advances in Analytical Approaches for Glycan and Glycopeptide Quantitation

“…As glycan permethylation (40) is routinely employed to reduce the high hydrophilicity of glycans and increase ionization efficiency prior to LC-MS analyses, early reports demonstrate simple workflow adaptation using iodomethane isotopologues to produce three labeling channels through light, medium, and heavy methyl labels (i.e., CH 3 , CD 2 H, CD 3 ) (41). The same research group later expanded this workflow into an eight-plex labeling strategy that included additional heavy carbon isotopes (42). Early reports of deuterium-based isotopic tags were provided by Bowman and Zaia, who first assessed multiple novel compounds for tetraplex labeling (43) and later applied them for glycan and glycosaminoglycan quantitation (44).…”

“…, CH 3 , CD 2 H, CD 3 ) ( 41 ). The same research group later expanded this workflow into an eight-plex labeling strategy that included additional heavy carbon isotopes ( 42 ). Early reports of deuterium-based isotopic tags were provided by Bowman and Zaia, who first assessed multiple novel compounds for tetraplex labeling ( 43 ) and later applied them for glycan and glycosaminoglycan quantitation ( 44 ).…”

Advances in mass spectrometry‐based glycomics—An update covering the period 2017–2021

“…Another way of introducing mass difference is isotopic permethylation. Mechref and coworkers [153] recently reported an eight-plex sample analysis, using CH 2 DI, CHD 2 I, CD 3 I, 13 CH 3 I, 13 CH 2 DI, 13 CHD 2 I, 13 CD 3 I, and CH 3 I. This method is capable of analyzing eight samples at a time, which dramatically shortened the analyzing time and minimized the ionization fluctuation caused by the different spray quality during ESI.…”

Separation of Permethylated O-Glycans, Free Oligosaccharides, and Glycosphingolipid-Glycans Using Porous Graphitized Carbon (PGC) Column

“…Figure 1 shows the separation of permethylated O-glycans released from k-casein, with insets depicting MS2 for each isomer. To reduce chromatographic bias, O-glycans were multiplexed [32] during the permethylation with 13 C-labeled methyl iodide, as shown in Figure 1a. Figure 1b depicts permethylated O-glycans treated with an exoglycosidase, α2-3 neumeridase, and permethylated with 12 C methyl iodide.…”