Advances in mass spectrometry‐based glycoproteomics: An update covering the period 2017–2021

Abstract

Protein glycosylation is one of the most common posttranslational modifications, and plays an essential role in a wide range of biological processes such as immune response, intercellular signaling, inflammation, host-pathogen interaction, and protein stability. Glycoproteomics is a proteomics subfield dedicated to identifying and characterizing the glycans and glycoproteins in a given cell or tissue. Aberrant glycosylation has been associated with various diseases such as Alzheimer's disease, viral infections, inflammation, immune deficiencies, congenital disorders, and cancers. However, glycoproteomic analysis remains challenging because of the low abundance, site-specific heterogeneity, and poor ionization efficiency of glycopeptides during LC-MS analyses. Therefore, the development of sensitive and accurate approaches to efficiently characterize protein glycosylation is crucial. Methods such as metabolic labeling, enrichment, and derivatization of glycopeptides, coupled with different mass spectrometry techniques and bioinformatics tools, have been developed to achieve sophisticated levels of quantitative and qualitative analyses of glycoproteins. This review attempts to update the recent developments in the field of glycoproteomics reported between 2017 and 2021.

Hydrophilic Peptide and Glycopeptide as Immobilized Sorbents for Glycosylation Analysis

“…To prevent ion suppression or ionization competition of the peptides, glycopeptides are usually enriched from peptides after proteolytic digestion. 8 The main glycopeptide enrichment methods vary depending on the glycopeptide of interest. For example, lectin affinity is specific to specific carbohydrate moieties, such as sialoglycoproteins or sialoglycopeptides, by SNA (black elderberry agglutinin) or MAL-II (Maackia Amurensis lectin II).…”

“…This is because peptides are more hydrophobic and are more easily ionized by MS. Additionally, the higher abundance of peptides when biological samples are digested with proteases can complicate the analysis of glycopeptides. To prevent ion suppression or ionization competition of the peptides, glycopeptides are usually enriched from peptides after proteolytic digestion . The main glycopeptide enrichment methods vary depending on the glycopeptide of interest.…”

Glycan/Protein-Stable Isotope Labeling in Cell Culture for Enabling Concurrent Quantitative Glycomics/Proteomics/Glycoproteomics

“…However, due to the glycan compositional and structural diversity, low abundance, and macro-/microheterogeneity, protein glycosylation analysis remains challenging. During the last few decades, a variety of mass spectrometry (MS)-based methods were developed to facilitate reliable characterization and quantitation of protein and protein glycosylation analysis. − …”

Isomeric Separation of α2,3/α2,6-Linked 2-Aminobenzamide (2AB)-Labeled Sialoglycopeptides by C18-LC-MS/MS

“…Liquid chromatography–mass spectrometry (LC–MS) offers a powerful approach to distinguish sialic acid linkage isomers. − In LC–MS-based glycomics analysis, linkage or positional isomers can be separated on released native or derivatized glycans. − Because the negative charge on sialic acid affects the ionization efficiency, a more common strategy to discriminate α2,3- and α2,6-linked sialic acid is to generate a mass difference of the isomers through chemical derivatizations. − Upon derivatization reactions, α2,6-linked sialic acids were converted to ester/amides, while α2,3-linked sialic acids yielded lactone/methylamide, which introduced a mass difference with high specificity. − However, isolated glycans exclude microheterogeneity of the glycosylation site. To obtain site-specific information, distinguishing isobaric glycoforms at the glycopeptide level is an emerging field of research.…”