Glycoproteomics is coming of age, thanks to advances in instrumentation, experimental methodologies and computational search algorithms.

Glycosylation is one of the most common post-translational modifications, and glycoproteins play crucial roles in important biological processes like cell signaling, host–pathogen interaction, immune response and disease, including cancer and even the ongoing COVID-19 pandemic (Science 369, 330–333, 2020). Glycoproteomics aims to determine the positions and identities of the complete repertoire of glycans and glycosylated proteins in a given cell or tissue.

Glycans are everywhere. High-throughput glycoproteomics approaches offer insights. Credit: Katherine Vicari, Springer Nature

Mass spectrometry (MS)-based approaches allow large-scale global analysis; however, the structural diversity of glycans and the heterogeneous nature of glycosylation sites make comprehensive analysis particularly challenging. Glycans obstruct complete fragmentation of the protein backbone, and they were traditionally removed for simplicity at the cost of losing glycan information. The MS spectra tend to be complicated due to the presence of isomers, often requiring manual interpretation. Furthermore, database searching for spectral matches can quickly become a combinatorial problem and requires innovative bioinformatics solutions.

Recent developments in MS instrumentation, fragmentation strategies (J. Proteome Res. 19, 3286–3301, 2020) and high-throughput workflows have made analyzing intact glycoproteins a possibility. Several specific enrichment strategies have made even low-abundance glycans and glycopeptides detectable (Mol. Cell. Proteomics, 2020). A variety of experimental workflows tailored for either N-linked glycans, which are found at consensus sites on the proteins, or O-linked glycans, which have no recognizable consensus sequence, have been developed (Nature 549, 538–542, 2017; Nat. Commun. 11, 5268, 2020; Nat. Methods 16, 902–910, 2019). New software packages based on fragment-ion indexing strategies offer substantial increases in speed for glycopeptide and site assignments (Nat. Methods 17, 1125–1132, 2020; Nat. Methods 17, 1133–1138, 2020).

With other -omics fields taking the lion’s share of attention in recent years, it is now time for glycoproteomics to shine. Comprehensive understanding of glycosylation at different levels of granularity is bound to serve both basic and translational research.

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Correspondence to Arunima Singh.

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Singh, A. Glycoproteomics. Nat Methods 18, 28 (2021).

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