Utility of Ion-Mobility Spectrometry for Deducing Branching of Multiply Charged Glycans and Glycopeptides in a High-Throughput Positive ion LC-FLR-IMS-MS Workflow

Abstract

High-throughput glycan analysis has become an important part of biopharmaceutical production and quality control. However, it is still a significant challenge in the field of glycomics to easily deduce isomeric glycan structures, especially in a highthroughput manner. Ion mobility spectrometry (IMS) is an excellent tool for differentiating isomeric glycan structures. However, demonstrations of the utility of IMS in high-throughput workflows such as liquid chromatography-fluorescence-mass spectrometry (LC-FLR-MS) workflows have been limited with only a small amount of collision cross section (CCS) data available. In particular, IMS data of glycan fragments obtained in positive ion mode are limited in comparison to those obtained in negative ion mode despite positive ion mode being widely used for glycomics. Here, we describe IMS TW CCSN 2 data obtained from a high-throughput LC-FLR-IMS-MS workflow in positive ion mode. We obtained IMS data from a selection of RapiFluor-MS (RFMS) labeled Nglycans and also glycopeptides. We describe how IMS is able to distinguish isomeric N-glycans and glycopeptides using both intact IMS and fragment-based IMS glycan sequencing experiments in positive ion mode, without significantly altering the high-throughput nature of the analysis. For the first time, we were able to successfully use IMS in positive ion mode to determine the branching of isomeric glycopeptides and RFMS labeled glycans. Further, we highlight that IMS glycan sequencing of fragments obtained from RFMS labeled glycans was similar to that of glycopeptides. Finally, we show that the IMS glycan sequencing approach can highlight shared structural features of nonisomeric glycans in a high-throughput LC-FLR-IMS-MS workflow.

High sensitivity glycomics in biomedicine

“…However, as opposed to CE, the analytes are ionized and interact with a carrier gas. The powerful technique has already been demonstrated for its ability to separate isomeric glycan species (Pallister et al, 2020; Sastre F I G U R E 6 Effect of different post-column makeup flow supplements on N-and O-glycan detection. The fold change of the AUC relative to the original setup is plotted with supplements consisting of methanol (orange), isopropanol (blue) or acetonitrile (green), resulting in a net concentration of 57% organic solvent (v/v) at the ion source.…”

A new approach for identifying positional isomers of glycans cleaved from monoclonal antibodies

“…[19][20][21][22] Several methods have been implemented to distinguish and identify positional isomers of released N-linked glycans with terminal Gal (α1-6/α1-3). These include tandem mass spectrometry, 23 ion mobility spectrometry (IMS), 24 and various combinations of selective enzymatic digestion or synthesis with nuclear magnetic resonance (NMR) or high-performance liquid chromatographic (HPLC) analysis. [25][26][27][28] The most commonly used method currently combines hydrophobic interaction liquid chromatography (HILIC) and mass spectrometry (MS), where the chromatographic peak assignment is based on the previously published work indicating that the glycan with a terminal galactose on the upper Man (α1-6) arm elutes prior to that with galactose on the lower Man (α1-3) arm.…”

“…Recently there has been a surge in the application of gasphase spectroscopy together with ion mobility spectrometry for the structural characterization of glycans. 24,[31][32][33][34][35][36] In the present work, we use a combination of ultrahigh-resolution IMS with cryogenic infrared spectroscopy as a rapid and reliable technique for glycan isomer identification. Our approach allows one to obtain highly resolved, isomer-specific vibrational spectra, even of larger, more complex glycan ions.…”

Semi-Automated Glycoproteomic Data Analysis of LC-MS Data Using GlycopeptideGraphMS in Process Development of Monoclonal Antibody Biologics

“…Electron transfer dissociation (ETD), for example, has been used effectively to identify the site of the glycan on the glycopeptide ( Riley and Coon, 2018 ). Additionally, ion mobility has recently been used to characterise glycan arm isomers ( Pallister et al, 2020 ). As one can appreciate, significant expertise and effort as well as advanced mass specrometers with higher specifications are often needed to resolve more information about a glyco-conjugates’ structure.…”