Advances in glycopeptide enrichment methods for the analysis of protein glycosylation over the past decade

Abstract

Advances in bioanalytical technology have accelerated the analysis of complex protein glycosylation, which is beneficial to understand glycosylation in drug discovery and disease diagnosis. Due to its biological uniqueness in the course of disease occurrence and development, disease‐specific glycosylation requires quantitative characterization of protein glycosylation. We provide a comprehensive review of recent advances in glycosylation analysis, including workflows for glycoprotein digestion, glycopeptide separation and enrichment, and mass spectrometry sequencing. We specifically focus on different strategies for glycopeptide enrichment through physical interaction, chemical oxidation, or metabolic labeling of intact glycopeptides. Recent advances and challenges of O‐glycosylation analysis are presented, and the development of improved enrichment methods combining different proteases to analyze O‐glycosylation is also proposed.

Recent development of analytical methods for disease-specific proteinO-GlcNAcylation

“…Periodate oxidation was most efficient on exocyclic and cis -diols; thus, N -glycopeptides are easily oxidized by sodium periodate via different monosaccharides (sialic acid, fucose, mannose, galactose, GalNAc and mannose). 39 Because GlcNAc contains trans -diol rather than cis -diol, the oxidation condition is more severe than for monosaccharide containing cis -diol. Oxidation of GlcNAc trans -diol at C3 and C4 requires higher temperatures and longer incubation times.…”

Recent advances in demystifying O‐glycosylation in health and disease

“…Different enrichment methods can be used depending on the research objective, including organic solvent precipitation, antibody capture, click chemistry enrichment, lectin affinity, hydrophilic interaction liquid chromatography (HILIC), chemoenzymatic and solid-phase chemoenzymatic methods [5,[46][47][48][49][50][51]. Acetone based solvent strategies can be used to extract O-glycopeptides, where the ratio of acetone to the sample organic solution depends on the type of glycopeptides, for example, 9:1(acetone to water) for O-glycopeptides and 5:1 for N-glycopeptides, even though the ratio of acetone to sample is less specific for extraction of different types of O-glycopeptides [52].…”

Recent development in hydrophilic interaction liquid chromatography stationary materials for glycopeptide analysis

“…The procedure typically includes protein extraction and digestion, glycopeptide enrichment, and mass spectrometry analysis. 42,100 Because it can universally enrich different types of glycopeptides, HILIC is widely used in sample preparation for mass spectrometry analysis of glycopeptides.…”