Analysis of N‐ and O‐linked site‐specific glycosylation by ion mobility mass spectrometry: State of the art and future directions

Glycosylation, the major post‐translational modification of proteins, significantly increases the diversity of proteoforms. Glycans are involved in a variety of pivotal structural and functional roles of proteins, and changes in glycosylation are profoundly connected to the progression of numerous diseases. Mass spectrometry (MS) has emerged as the gold standard for glycan and glycopeptide analysis because of its high sensitivity and the wealth of fragmentation information that can be obtained. Various separation techniques have been employed to resolve glycan and glycopeptide isomers at the front end of the MS. However, differentiating structures of isobaric and isomeric glycopeptides constitutes a challenge in MS‐based characterization. Many reports described the use of various ion mobility–mass spectrometry (IM–MS) techniques for glycomic analyses. Nevertheless, very few studies have focused on N‐ and O‐linked site‐specific glycopeptidomic analysis. Unlike glycomics, glycoproteomics presents a multitude of inherent challenges in microheterogeneity, which are further exacerbated by the lack of dedicated bioinformatics tools. In this review, we cover recent advances made towards the growing field of site‐specific glycosylation analysis using IM–MS with a specific emphasis on the MS techniques and capabilities in resolving isomeric peptidoglycan structures. Furthermore, we discuss commonly used software that supports IM–MS data analysis of glycopeptides.