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Large-scale identification of ubiquitination sites by mass spectrometry

Nature Protocols volume 8, pages 1950–1960 (2013)Cite this article

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Abstract

Ubiquitination is essential for the regulation of cellular protein homeostasis. It also has a central role in numerous signaling events. Recent advances in the production and availability of antibodies that recognize the Lys-ɛ-Gly-Gly (K-ɛ-GG) remnant produced by trypsin digestion of proteins having ubiquitinated lysine side chains have markedly improved the ability to enrich and detect endogenous ubiquitination sites by mass spectrometry (MS). The following protocol describes the steps required to complete a large-scale ubiquitin experiment for the detection of tens of thousands of distinct ubiquitination sites from cell lines or tissue samples. Specifically, we present detailed, step-by-step instructions for sample preparation, off-line fractionation by reversed-phase chromatography at pH 10, immobilization of an antibody specific to K-ɛ-GG to beads by chemical cross-linking, enrichment of ubiquitinated peptides using these antibodies and proteomic analysis of enriched samples by LC–tandem MS (MS/MS). Relative quantification can be achieved by performing stable isotope labeling by amino acids in cell culture (SILAC) labeling of cells. After cell or tissue samples have been prepared for lysis, the described protocol can be completed in ∼5 d.

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Figure 1: Enrichment of K-ɛ-GG peptides using anti–K-ɛ-GG antibody.
Figure 2: Workflow for preparing samples for K-ɛ-GG enrichment.
Figure 3: Example of an SDS-PAGE gel used to evaluate the efficiency of antibody cross-linking to protein A beads.
Figure 4: Analysis of K-ɛ-GG peptide data.
Figure 5: MS/MS spectrum of a K-ɛ-GG peptide.

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Acknowledgements

We thank L. Gaffney for help with illustrations. This work was supported in part by the Broad Institute of MIT and Harvard and by grants from the US National Cancer Institute (U24CA160034, part of the Clinical Proteomics Tumor Analysis Consortium initiative; to S.A.C.) and the National Heart, Lung and Blood Institute (HHSN268201000033C and R01HL096738; to S.A.C.).

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  1. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    Namrata D Udeshi, Philipp Mertins, Tanya Svinkina & Steven A Carr

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  1. Namrata D Udeshi
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Contributions

N.D.U., P.M., T.S. and S.A.C. developed the protocol. N.D.U. and S.A.C. wrote the manuscript with input from all authors.

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Correspondence to Namrata D Udeshi or Steven A Carr.

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Udeshi, N., Mertins, P., Svinkina, T. et al. Large-scale identification of ubiquitination sites by mass spectrometry. Nat Protoc 8, 1950–1960 (2013). https://doi.org/10.1038/nprot.2013.120

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