A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics

Abstract

Glycosylation is a prevalent, yet heterogeneous modification with a broad range of implications in molecular biology. This heterogeneity precludes enrichment strategies that can be universally beneficial for all glycan classes. Thus, choice of enrichment strategy has profound implications on experimental outcomes. Here we review common enrichment strategies used in modern mass spectrometry (MS)-based glycoproteomic experiments, including lectins and other affinity chromatographies, hydrophilic interaction chromatography (HILIC) and its derivatives, porous graphitic carbon (PGC), reversible and irreversible chemical coupling strategies, and chemical biology tools that often leverage bioorthogonal handles. Interest in glycoproteomics continues to surge as MS instrumentation and software improve, so this review aims to help equip researchers with necessary information to choose appropriate enrichment strategies that best complement these efforts.

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

“…To calibrate the MS and trapped ion mobility spectrometry (TIMS) device, Agilent tune mix was directly infused to provide species of known mass and reduced mobility. 37 For MS calibration, the MS resolution for the most abundant calibrant signal, 1222 m/z, was 62,000. Calibrant points at 922, 1222, and 1522 m/z were used for TIMS calibration.…”

“…Interestingly, these two Core 1 O-glycans were previously reported for the S-RBD as potential modifications, however the previous studies were not able to resolve the exact glycoforms due to the challenges arising from inferring intact glycoprotein structures from peptide digests and the signal low abundance of O-glycans under conventional MS analysis. 22,23,37 However, our native top-down MS strategy enables us to unambiguously reveal the exact glycoform corresponding to each of these Core 1 O-glycans. Two additional Core 2 glycan structures were identified GalNAcGal(GalNeuAc)(GlcNAcGal) (15+ most abundant charge state, centered at 1748.1 m/z, with additional N-terminal acetylated proteoform found at 1750.5 m/z) (Figures S9,10) and GalNAcGal(GalNeuAc)(GlcNAcGalNeuAc) (centered at 1767.6 m/z) (Figure S11).…”

“…28,[34][35][36] Conventional methods to analyze protein glycosylation rely on the bottom-up MS approach of analyzing glycopeptides obtained through enzymatic digestion. 31,37,38 Although the bottom-up MS approach is a high throughput method for global proteome identification of glycosites and Nglycan heterogeneity from complex samples, the relative abundance of various intact glycoforms and the overall post-translational modification (PTM) compositions of different co-appearing 'proteoforms' 39 are lost. [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.…”

Towards structure-focused glycoproteomics

“…The enrichment methods used in glycoproteomics have been comprehensively reviewed [ 135 ]. While some enrichment strategies including HILIC-SPE aim to capture the entire complement of glycopeptides to provide a quantitative view of the glycoproteome [ 80 ], structure-specific enrichment approaches aim to capture only a subset of glycopeptides carrying specific glycan structures.…”

Revealing the human mucinome

“…We reasoned that the catalytically inactive point mutant of StcE (StcE E447D ) could function as a universal mucin enrichment tool for mucin domain discovery, similar to how O-glycosidases and engineered sialidases can enrich broadly for O-glycosylated and sialylated glycoproteins, respectively 44,45 . Here we show that StcE E447D -conjugated beads selectively enrich mucin-domain glycoproteins from complex cancer cell lysates and from crude ovarian cancer patient ascites fluid.…”