Recent trends in glycoproteomics by characterization of intact glycopeptides

Abstract

This trends article provides an overview of the state of the art in the analysis of intact glycopeptides by proteomics technologies based on LC-MS analysis. A brief description of the main techniques used at the different steps of the analytical workflow is provided, giving special attention to the most recent developments. The topics discussed include the need for dedicated sample preparation for intact glycopeptide purification from complex biological matrices. This section covers the common approaches with a special description of new materials and innovative reversible chemical derivatization strategies, specifically devised for intact glycopeptide analysis or dual enrichment of glycosylation and other post-translational modifications. The approaches are described for the characterization of intact glycopeptide structures by LC-MS and data analysis by bioinformatics for spectra annotation. The last section covers the open challenges in the field of intact glycopeptide analysis. These challenges include the need of a detailed description of the glycopeptide isomerism, the issues with quantitative analysis, and the lack of analytical methods for the large-scale characterization of glycosylation types that remain poorly characterized, such as C-mannosylation and tyrosine O-glycosylation. This bird's-eye view article provides both a state of the art in the field of intact glycopeptide analysis and open challenges to prompt future research on the topic.

Keywords: Enrichment; Glycoproteomics; Intact glycopeptides; Protein glycosylation; Qualitative analysis; Quantitative analysis.

Fig. 1

Examples of mammalian N-glycosylations (a), O-glycosylations (b), C-glycosylation (c), with explanation of glycan graphical representation (d). Amino acid side chains are shown boxed. Common sequons are also shown, with green residues to mark the glycosite. Structures were drawn using GlycanBuilder2 [3]

Fig. 2

Number of published papers dealing with N-, O-, or C-glycosylation over the last years and resulting in Scopus database accessed in December 2022

Fig. 3

Graphic representation of the enrichment mechanism of sialylated glycopeptides by reversible binding. The support (red) has a linked glucose moiety through Schiff base (yellow). This material (1) can enrich sialylated glycopeptides (2) by hydrolysis of the Schiff base due to reaction with the sialic acid (blue) in the glycopeptide (purple). The hydrolysis of the Schiff base releases the linked glucose (3) and results in the formation of a complex (4) between the protonated amine of the stationary phase and the sialylated glycan. The complex is finally disrupted by ammonia upon elution and releases the support (5) and the intact sialylated glycopeptide (2)

Fig. 4

Reversible chemical derivatization for the enrichment of N-linked glycopeptides (a) and O-GlcNacetylation glycopeptides (b) using hydrazine chemistry