Accurate Identification of Isomeric Glycans by Trapped Ion Mobility Spectrometry-Electronic Excitation Dissociation Tandem Mass Spectrometry

Abstract

Ion mobility-mass spectrometry (IM-MS) has become a powerful tool for glycan structural characterization due to its ability to separate isomers and provide collision cross section (CCS) values that facilitate structural assignment. However, IM-based isomer analysis may be complicated by the presence of multiple gas-phase conformations of a single structure that not only increases difficulty in isomer separation but can also introduce the possibility for misinterpretation of conformers as isomers. Here, the ion mobility behavior of several sets of isomeric glycans, analyzed as their permethylated derivatives, in both non-reduced and reduced forms, was investigated by gated-trapped ion mobility spectrometry (G-TIMS). Notably, reducingend reduction, commonly performed to remove anomerism-induced chromatographic peak splitting, did not eliminate the conformational heterogeneity of permethylated glycans in the gas phase. At a mobility resolving power of ~100, 14 out of 22 structures showed more than one conformation. These results highlight the need to use IMS devices with high mobility resolving power for better separation of isomers and to acquire additional structural information that can differentiate isomers from conformers. On-line electronic excitation dissociation (EED) MS/MS analysis of isomeric glycan mixtures following G-TIMS separation showed that EED can generate isomer-specific fragments while producing nearly identical tandem mass spectra for conformers, thus allowing confident identification of isomers with minimal evidence of any ambiguity resulting from the presence of conformers. G-TIMS EED MS/MS analysis of N-linked glycans released from ovalbumin revealed that several mobility features previously thought to arise from isomeric structures were conformers of a single structure. Finally, analysis of ovalbumin N-*

Identification of N-glycan positional isomers by combining IMS and vibrational fingerprinting of structurally determinant CID fragments

“…21 The use of various IMS technologies together with tandem MS has been reported for the analysis of isomeric glycans, including positional isomers. [22][23][24][25][26][27][28][29][30][31][32] While ultrahigh-resolution IMS based on structures for lossless ion manipulation (SLIM) 33,34 has been applied to achieve separation of different kinds of glycan isomers, 35,36 the assignment of a drift peak to a precise isomeric form often remains elusive. 35,37 We have previously demonstrated that cryogenic vibrational spectroscopy can be used for the identification of glycan isomers based on their unique infrared (IR) absorption spectrum.…”

Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry

“…[40][41][42] In contrast, by combining glycoproteomics with the top-down MS approach, which preserves the intact glycoprotein enabling high-resolution proteoform-resolved analysis, [43][44][45] we could achieve the simultaneous characterization of the molecular structures, the site specificity, and the relative abundance of various glycoforms. Furthermore, by integrating native MS, which has recently emerged as a powerful structural biology tool to study protein structure-function relationships, [46][47][48][49][50][51][52] with trapped ion mobility spectrometry (TIMS), [53][54][55] we can investigate the gas phase structural variants to achieve the direct quantification of individual glycoproteoforms.…”

Identification of N-glycans with GalNAc-containing antennae from recombinant HIV trimers by ion mobility and negative ion fragmentation

“…The masses of these ions showed that the location of the GalNAc residue in glycans with one GalNAc was invariably on the 3-antenna, a property that would be difficult to determine by traditional positive ion fragmentation methods. However, recent methods such as electronic excitation dissociation (EED) do appear to produce ions that allow this structural feature to be determined directly [46]. The current paper, therefore, provides a further illustration of how ion mobility and negative ion CID, combined with exoglycosidase digestions to determine the nature of the monosaccharide constituents, can be used to provide detailed structures of these compounds without the need for extensive fractionation or derivatization.…”