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Proteomic strategies for characterizing ubiquitin-like modifications

Abstract

The modification of proteins by the addition of ubiquitin and other ubiquitin-like proteins (UBLs) is involved in a wide range of cellular processes including cell cycle progression, the DNA damage response, endocytosis, cell signalling, autophagy and protein quality control. The UBL family comprises more than a dozen structurally related members, with ubiquitin, small ubiquitin-like modifier (SUMO) proteins, NEDD8, ISG15 and FAT10 being the most commonly known. Each UBL is associated with a distinct set of enzymes that alter the architecture and fate of their cognate proteins. UBL-conjugating enzymes add one or more UBLs to lysine and non-lysine acceptor sites on their target proteins, forming a complex distribution of monomeric and polymeric modifications. Different approaches and strategies are available to identify the sites of UBL modification, the types of modification and their dynamics upon various cellular stimuli; these techniques can decipher the complex architecture of UBL substrates and expand our understanding of UBL functions and their importance in cellular homeostasis and human diseases. This Primer covers the current methods for identifying UBL substrates, their modification sites and UBL chain linkages, and describes where the application of these methods can be used to gain biological insights into UBL functions.

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Fig. 1: Overview of the UBL conjugation machinery and information available from UBL proteomics experiments.
Fig. 2: Identification of targets of UBLs using strategies that exogenously express UBLs or related enzymes.
Fig. 3: Identification of ubiquitin or UBLs using strategies that enrich endogenous UBL proteins.
Fig. 4: Quantitative proteomic strategies applied to UBL proteomics.
Fig. 5: Data analysis workflow and example of MS/MS spectra.
Fig. 6: Crosstalk between ubiquitin and UBLs.
Fig. 7: Roles of UBL modifications in the cell.

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Acknowledgements

This work was carried out with financial support from the Natural Sciences and Engineering Research Council (NSERC 311598). IRIC proteomics facility is a Genomics Technology platform funded in part by the Canadian Government through Genome Canada, the Canadian Center of Excellence in Commercialization and Research, and the Canadian Foundation for Innovation. A.C.O.V. is supported by the European Research Council, the Dutch Research Council (NWO) and the Dutch Cancer Society.

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Authors and Affiliations

Authors

Contributions

Introduction (P.T.); Experimentation (C.L., T.G.N., P.T. and A.C.O.V.); Results (A.C.O.V.); Applications (P.T.); Reproducibility and data deposition (A.C.O.V.); Limitations and optimizations (A.C.O.V.); Outlook (A.C.O.V.); Figs 1, 2, 3 and 5 (C.L.); Figs 4 and 7 and Box 1 and 2 (C.L. and T.G.N.); Table 1 (C.L., T.G.N. and P.T.); Overview of the Primer (A.C.O.V. and P.T.). All authors reviewed and edited the final manuscript.

Corresponding authors

Correspondence to Alfred C. O. Vertegaal or Pierre Thibault.

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All authors declare no competing interests.

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Peer review information

Nature Reviews Methods Primers thanks A. Ordureau, J. Peng, A. Pinto-Fernandez, G. Vere and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Biorender: https://biorender.com/

iProX: https://www.iprox.org/

Japan ProteOme Standard Repository/Database: https://jpostdb.org/

JASSA: http://www.jassa.fr

Mass Spectrometry Interactive Virtual Environment (MassIVE): https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp

Panorama Public: https://panoramaweb.org

PeptideAtlas: http://www.peptideatlas.org/

PhosphoSitePlus: https://www.phosphosite.org

PRoteomics IDEntifications Database (PRIDE): https://www.ebi.ac.uk/pride/

UniProt: https://www.uniprot.org

Glossary

Ubiquitin code

The concept that distinct conformations of ubiquitin chains and modifications lead to different cellular outcomes.

De-NEDDylation

Removal of NEDD8 from modified substrates.

Isopycnic centrifugation

A fractionation method where cell components can be separated based on density gradient centrifugation.

Remnant peptides

Amino acids left over on modified lysine residues after proteolytic digestion.

Ubiquitylome

The cell-wide repertoire of ubiquitylated proteins.

Offline fractionation

Fractionation of peptide extracts by methods such as ion exchange or high-pH, reverse-phase chromatography; fractions are subsequently analysed by mass spectrometry.

Head-to-tail concatemers

Long, continuous DNA molecules containing multiple copies of the same gene assembled head to tail.

UBL traps

Affinity purification methods where fusion proteins containing units of ubiquitin-like protein (UBL)-binding domains are expressed in cells to trap UBL conjugates.

Ubiquitin clipping

A technique that uses an engineered viral protease, Lbpro, to cleave ubiquitin conjugates and leave a traceable diglycine remnant on the modified substrates.

Pulse chase

An experiment where cells are exposed to a labelled compound that is incorporated into proteins, which is later replaced with an unlabelled form to determine the time of exchange.

Ratio compression

Underestimation of the fold-change ratio of peptide/protein abundance in isobaric peptide labelling, owing to co-selection of different peptides during tandem mass spectrometry (MS/MS).

Translesion synthesis

A process whereby the DNA replication machinery can bypass the blocked replication fork caused by DNA damage.

Affimers

Non-antibody binding proteins that mimic the molecular recognition features of antibodies.

Top-down proteomics

A protein identification method that relies on the selection of protein ions as precursors for tandem mass spectrometry (MS/MS) fragmentation.

Proteolysis-targeting chimaeras

(PROTACs). Heterobifunctional small molecules that consist of a ubiquitin E3 ligase-binding domain linked to a domain that bind specifically to a protein targeted for degradation.

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Li, C., Nelson, T.G., Vertegaal, A.C.O. et al. Proteomic strategies for characterizing ubiquitin-like modifications. Nat Rev Methods Primers 1, 53 (2021). https://doi.org/10.1038/s43586-021-00048-9

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