Abstract
Protein glycosylation involves the co-translational or post-translational addition of glycans to proteins and is a crucial protein modification in health and disease. The aim of glycoproteomics is to understand how glycosylation shapes biological processes by understanding peptide sequences, glycan structures and sites of modification in a system-wide context. Over the past two decades, mass spectrometry (MS) has emerged as the primary technique for studying glycoproteins, with intact glycopeptide analysis — the study of glycopeptides decorated with their native glycan structures — now a preferred approach across the community. In this Primer, we discuss glycoproteomic methods for studying glycosylation classes, including best practices and critical considerations. We summarize how glycoproteomics is used to understand glycosylation at a systems level, with a specific focus on N-linked and O-linked glycosylation (both mucin-type and O-GlcNAcylation). We cover topics that include sample selection; techniques for protein isolation, proteolytic digestion, glycopeptide enrichment and MS fragmentation; bioinformatic platforms and applications of glycoproteomics. Finally, we give a perspective on where the field is heading. Overall, this Primer outlines the current technologies, persistent challenges and recent advances in the exciting field of glycoproteomics.
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Acknowledgements
N.E.S. is supported by an Australian Research Council Future Fellowship (FT200100270). B.L.P. is supported by an Australian National Health and Medical Research Emerging Leader Grant (APP 2009642). M.T.-A. is supported by an Australian Research Council Future Fellowship (FT210100455). D.A.P. and A.I.N. are supported by NIH grants R01-GM-094231 and U24-CA210967. S.A.M. is supported by the Yale Science Development Fund and the Yale SEAS/Science Program to Advance Research Collaboration (SPARC). A.H. is supported by a research grant (00025438) from VILLUM FONDEN. K.S. is supported by an Excellence grant from the Novo Nordisk Foundation (NNF17OC0026030). H.H.W. is supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (GlycoSkin H2020-ERC; 772735), Lundbeck Foundation (R313-2019-869) and the Neye Foundation. The Copenhagen Center for Glycomics including I.B., K.S., A.H., H.H.W. and S.Y.V. are supported by a Danish National Research Foundation grant (DNRF107). N.M.R. acknowledges support from a NIH Predoctoral to Postdoctoral Transition award (K00 CA212454). K.F.A.-K. is supported by the Japan Science and Technology Agency (JST) and National Bioscience Database Center (NBDC) of Japan, grant number 17934031.
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Contributions
Introduction (B.L.P. and M.T.-A.), Experimentation (I.B., S.A.M., D.A.P., N.M.R., K.S., A.I.N., C.R.B. and H.H.W.), Results (S.Y.V., A.H., D.A.P. and S.A.M.), Applications (I.B., K.S., C.R.B. and H.H.W.), Reproducibility and data deposition (K.F.A.-K. and M.T.-A.), Limitations and optimizations (N.E.S.), Outlook (N.E.S. and B.L.P.). Overview of the Primer (N.E.S.).
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Nature Reviews Methods Primers thanks Yehia Mechref and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Reporting guidelines for glycomics: https://www.beilstein-institut.de/en/projects/mirage/guidelines
Glossary
- Non-templated
-
A process that is not guided by a template, in contrast to templated processes such as DNA transcription and translation.
- Anomeric linkages
-
A linkage description that denotes the connectivity of one monosaccharide to another relative to the anomeric centre of the monosaccharide.
- Intact glycopeptides
-
Glycopeptides decorated with their native or near-native glycoforms.
- Bottom-up glycoproteomics
-
The analysis of glycoproteome samples based on the identification of proteolytically generated glycopeptides.
- Proteoforms
-
A proteoform is a specific form of a protein defined by its exact amino acid sequence and post-translational modification status at specific residues in the protein.
- Dynamic range
-
Within the context of bottom-up glycoproteomics, the range of quantifiable ion intensities between the most abundant observable glycopeptide and the least abundant glycopeptide.
- Glycoforms
-
Different molecular forms of a glycoprotein for which either the glycan composition or site of attachment can differ from another glycosylated form of the protein.
- Chaotropic agents
-
Agents that cause perturbations in non-covalent forces, leading to the disruption of molecular structures aiding in the solubilization of proteins.
- Positive polarity mode
-
A mode of operation of a mass spectrometer conducive to the analysis of positively charged ions.
- Proteome coverage
-
The depth of analysis achieved for a given proteome experiment, referring to the total number of protein identifications or the average number of peptides or sequence coverage identified per protein.
- Exoprotease
-
A protease that catalyses the removal of N-terminal or C-terminal amino acids from a protein sequence.
- Endoprotease
-
A protease that catalyses the cleavage of amino acids within a protein sequence.
- Non-reducing termini
-
The termini of a carbohydrate chain that are unable to initiate a reduction reaction.
- Lectin
-
A general term given to describe carbohydrate-binding proteins, which typically have affinity for specific sugars and modest binding affinities.
- Click chemistry
-
A loosely defined set of chemical conjugation reactions that are typically highly efficient and occur between small chemical groups such as alkyne and azide groups.
- Bump-and-hole strategy
-
A chemical biology approach that involves the modification of enzymes to accommodate a non-native small molecule, such as a nucleotide-linked carbohydrate containing a chemical handle, enabling enrichment or imaging.
- Isomeric glycoforms
-
Glycans that have the same elemental composition but differ in monosaccharide arrangement; also isobaric in nature by definition.
- Online glycopeptide separation
-
The separation of glycopeptides or peptides using chromatographic approaches whereby the eluting samples are directly introduced into a mass spectrometer.
- Offline separation
-
Separations of glycopeptides or peptides using chromatographic approaches not directly interfaced with a mass spectrometer.
- Cross-ring fragments
-
Fragment ions that result from cleavage across a carbohydrate ring.
- Secondary gas-phase structure effects
-
The reduced efficiency of fragmentation caused by the formation of secondary structure within the gas phase of a polypeptide backbone.
- Isotopologues
-
Chemical species that differ in the number of neutrons resulting from the incorporation of stable isotopes such as 13C, 15N, 18O or 2H; these chemical species have near-identical physicochemical properties, but are observed with a heavier mass using mass spectrometry
- Sequon
-
A sequence (motif) of amino acids that corresponds to a predictable site of glycosylation.
- Top-down glycoproteomics
-
The analysis of glycoproteome samples based on intact, undigested glycoprotein species.
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Bagdonaite, I., Malaker, S.A., Polasky, D.A. et al. Glycoproteomics. Nat Rev Methods Primers 2, 48 (2022). https://doi.org/10.1038/s43586-022-00128-4
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DOI: https://doi.org/10.1038/s43586-022-00128-4
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