Research
A Proteome-wide, Quantitative Survey of In Vivo Ubiquitylation Sites Reveals Widespread Regulatory Roles*

https://doi.org/10.1074/mcp.M111.013284Get rights and content
Under a Creative Commons license
open access

Post-translational modification of proteins by ubiquitin is a fundamentally important regulatory mechanism. However, proteome-wide analysis of endogenous ubiquitylation remains a challenging task, and almost always has relied on cells expressing affinity tagged ubiquitin. Here we combine single-step immunoenrichment of ubiquitylated peptides with peptide fractionation and high-resolution mass spectrometry to investigate endogenous ubiquitylation sites. We precisely map 11,054 endogenous putative ubiquitylation sites (diglycine-modified lysines) on 4,273 human proteins. The presented data set covers 67% of the known ubiquitylation sites and contains 10,254 novel sites on proteins with diverse cellular functions including cell signaling, receptor endocytosis, DNA replication, DNA damage repair, and cell cycle progression. Our method enables site-specific quantification of ubiquitylation in response to cellular perturbations and is applicable to any cell type or tissue. Global quantification of ubiquitylation in cells treated with the proteasome inhibitor MG-132 discovers sites that are involved in proteasomal degradation, and suggests a nonproteasomal function for almost half of all sites. Surprisingly, ubiquitylation of about 15% of sites decreased more than twofold within four hours of MG-132 treatment, showing that inhibition of proteasomal function can dramatically reduce ubiquitylation on many sites with non-proteasomal functions. Comparison of ubiquitylation sites with acetylation sites reveals an extensive overlap between the lysine residues targeted by these two modifications. However, the crosstalk between these two post-translational modifications is significantly less frequent on sites that show increased ubiquitylation upon proteasome inhibition. Taken together, we report the largest site-specific ubiquitylation dataset in human cells, and for the first time demonstrate proteome-wide, site-specific quantification of endogenous putative ubiquitylation sites.

Cited by (0)

Author's Choice—Final version full access.

*

This work is supported by the European Commission's 7th Framework Programme grants Proteomics Research Infrastructure Maximizing knowledge EXchange and access (XS) (INFRASTRUCTURES-F7-2010-262067/PRIME-XS) and Systems Biology of Stem Cells and Reprogramming (HEALTH-F7-2010-242129/SYBOSS), and the Lundbeck Foundation (R48-A4649). SAW is supported by a postdoctoral grant from Danish Council for Independent Research (FSS: 10-085134). The Center for Protein Research is funded by a generous grant from the Novo Nordisk Foundation.

This article contains supplemental Figs. S1 to S3 and Tables S1 to S3.

1

The abbreviations used are:

    PTM

    post-translational modification

    UPS

    ubiquitin proteasome system

    SILAC

    stable isotope labeling by amino acids in cell culture

    HCD

    higher-energy C-trap dissociation

    GO

    gene ontology

    RTK

    receptor tyrosine kinase

    di-Gly

    di-glycine.

These authors contributed equally to this work.