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Refined Preparation and Use of Anti-diglycine Remnant (K-ε-GG) Antibody Enables Routine Quantification of 10,000s of Ubiquitination Sites in Single Proteomics Experiments*

  • Namrata D. Udeshi
    Correspondence
    To whom correspondence should be addressed: Steven A. Carr,Broad Institute of Massachusetts Institute of Technology and Harvard University, 7 Cambridge Center, Cambridge, MA 02142, Tel.:617-714-7630, Fax:617-714-8957, E-mail: [email protected]
    Affiliations
    Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts 02142
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  • Tanya Svinkina
    Affiliations
    Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts 02142
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  • Philipp Mertins
    Affiliations
    Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts 02142
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  • Eric Kuhn
    Affiliations
    Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts 02142
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  • D.R. Mani
    Affiliations
    Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts 02142
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  • Jana W. Qiao
    Affiliations
    Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts 02142
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  • Steven A. Carr
    Correspondence
    Namrata D. UdeshiBroad Institute of Massachusetts Institute of Technology and Harvard University, 7 Cambridge Center, Cambridge, MA 02142, Tel.:617-714-8322, Fax:617-714-8957, E-mail: [email protected]
    Affiliations
    Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts 02142
    Search for articles by this author
  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant U24CA160034 from NCI Clinical Proteomics Tumor Analysis Consortium Initiative (to S. A. C.) and Grants HHSN268201000033C and R01HL096738 from NHLBI (to S. A. C.). This work was also supported by the Broad Institute of Massachusetts Institute of Technology and Harvard University.
    This article contains supplemental material.
Open AccessPublished:December 24, 2012DOI:https://doi.org/10.1074/mcp.O112.027094
      Detection of endogenous ubiquitination sites by mass spectrometry has dramatically improved with the commercialization of anti-di-glycine remnant (K-ε-GG) antibodies. Here, we describe a number of improvements to the K-ε-GG enrichment workflow, including optimized antibody and peptide input requirements, antibody cross-linking, and improved off-line fractionation prior to enrichment. This refined and practical workflow enables routine identification and quantification of ∼20,000 distinct endogenous ubiquitination sites in a single SILAC experiment using moderate amounts of protein input.
      The commercialization of antibodies that recognize lysine residues modified with a di-glycine remnant (K-ε-GG)
      The abbreviations used are:
      K-ε-GG
      di-glycine remnant
      SILAC
      stable isotope labeling by amino acids in cell culture
      DMP
      dimethyl pimelimidate
      basic RP
      basic pH reversed phase
      HCD
      high energy collision-induced dissociation
      FA
      formic acid.
      1The abbreviations used are:K-ε-GG
      di-glycine remnant
      SILAC
      stable isotope labeling by amino acids in cell culture
      DMP
      dimethyl pimelimidate
      basic RP
      basic pH reversed phase
      HCD
      high energy collision-induced dissociation
      FA
      formic acid.
      has significantly transformed the detection of endogenous protein ubiquitination sites by mass spectrometry (
      • Emanuele M.J.
      • Elia A.E.
      • Xu Q.
      • Thoma C.R.
      • Izhar L.
      • Leng Y.
      • Guo A.
      • Chen Y.N.
      • Rush J.
      • Hsu P.W.
      • Yen H.C.
      • Elledge S.J.
      Global identification of modular Cullin-RING ligase substrates.
      ,
      • Wagner S.A.
      • Beli P.
      • Weinert B.T.
      • Nielsen M.L.
      • Cox J.
      • Mann M.
      • Choudhary C.
      A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles.
      ,
      • Udeshi N.D.
      • Mani D.R.
      • Eisenhaure T.
      • Mertins P.
      • Jaffe J.D.
      • Clauser K.R.
      • Hacohen N.
      • Carr S.A.
      Methods for quantification of in vivo changes in protein ubiquitination following proteasome and deubiquitinase inhibition.
      ,
      • Xu G.
      • Paige J.S.
      • Jaffrey S.R.
      Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling.
      ,
      • Kim W.
      • Bennett E.J.
      • Huttlin E.L.
      • Guo A.
      • Li J.
      • Possemato A.
      • Sowa M.E.
      • Rad R.
      • Rush J.
      • Comb M.J.
      • Harper J.W.
      • Gygi S.P.
      Systematic and quantitative assessment of the ubiquitin-modified proteome.
      ). Prior to the development of these highly specific reagents, proteomics experiments were limited to identification of up to only several hundred ubiquitination sites, which severely limited the scope of global ubiquitination studies (
      • Danielsen J.M.
      • Sylvestersen K.B.
      • Bekker-Jensen S.
      • Szklarczyk D.
      • Poulsen J.W.
      • Horn H.
      • Jensen L.J.
      • Mailand N.
      • Nielsen M.L.
      Mass spectrometric analysis of lysine ubiquitylation reveals promiscuity at site level.
      ). Recent proteomic studies employing anti-K-ε-GG antibodies have enhanced our understanding of ubiquitin biology through the identification of thousands of ubiquitination sites and the analysis of the change in relative abundance of these sites after chemical or biological perturbation (
      • Emanuele M.J.
      • Elia A.E.
      • Xu Q.
      • Thoma C.R.
      • Izhar L.
      • Leng Y.
      • Guo A.
      • Chen Y.N.
      • Rush J.
      • Hsu P.W.
      • Yen H.C.
      • Elledge S.J.
      Global identification of modular Cullin-RING ligase substrates.
      ,
      • Wagner S.A.
      • Beli P.
      • Weinert B.T.
      • Nielsen M.L.
      • Cox J.
      • Mann M.
      • Choudhary C.
      A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles.
      ,
      • Udeshi N.D.
      • Mani D.R.
      • Eisenhaure T.
      • Mertins P.
      • Jaffe J.D.
      • Clauser K.R.
      • Hacohen N.
      • Carr S.A.
      Methods for quantification of in vivo changes in protein ubiquitination following proteasome and deubiquitinase inhibition.
      ,
      • Kim W.
      • Bennett E.J.
      • Huttlin E.L.
      • Guo A.
      • Li J.
      • Possemato A.
      • Sowa M.E.
      • Rad R.
      • Rush J.
      • Comb M.J.
      • Harper J.W.
      • Gygi S.P.
      Systematic and quantitative assessment of the ubiquitin-modified proteome.
      ,
      • Ong S.-E.
      • Blagoev B.
      • Kratchmarova I.
      • Kristensen D.B.
      • Steen H.
      • Pandey A.
      • Mann M.
      Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.
      ). Use of stable isotope labeling by amino acids in cell culture (SILAC) for quantification has enabled researchers to better understand the extent of ubiquitin regulation upon proteasome inhibition and precisely identify those protein classes, such as newly synthesized proteins or chromatin-related proteins, that see overt changes in their ubiquitination levels upon drug treatment (
      • Wagner S.A.
      • Beli P.
      • Weinert B.T.
      • Nielsen M.L.
      • Cox J.
      • Mann M.
      • Choudhary C.
      A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles.
      ,
      • Udeshi N.D.
      • Mani D.R.
      • Eisenhaure T.
      • Mertins P.
      • Jaffe J.D.
      • Clauser K.R.
      • Hacohen N.
      • Carr S.A.
      Methods for quantification of in vivo changes in protein ubiquitination following proteasome and deubiquitinase inhibition.
      ,
      • Kim W.
      • Bennett E.J.
      • Huttlin E.L.
      • Guo A.
      • Li J.
      • Possemato A.
      • Sowa M.E.
      • Rad R.
      • Rush J.
      • Comb M.J.
      • Harper J.W.
      • Gygi S.P.
      Systematic and quantitative assessment of the ubiquitin-modified proteome.
      ). Emanuel et al. (
      • Emanuele M.J.
      • Elia A.E.
      • Xu Q.
      • Thoma C.R.
      • Izhar L.
      • Leng Y.
      • Guo A.
      • Chen Y.N.
      • Rush J.
      • Hsu P.W.
      • Yen H.C.
      • Elledge S.J.
      Global identification of modular Cullin-RING ligase substrates.
      ) have combined genetic and proteomics assays implementing the anti-K-ε-GG antibody to identify hundreds of known and putative Cullin-RING ligase substrates, which has clearly demonstrated the extensive role of Cullin-RING ligase ubiquitination on cellular protein regulation.
      Despite the successes recently achieved with the use of the anti-K-ε-GG antibody, increased sample input (up to ∼35 mg) and/or the completion of numerous experimental replicates have been necessary to achieve large numbers of K-ε-GG sites (>5,000) in a single SILAC-based experiment (
      • Emanuele M.J.
      • Elia A.E.
      • Xu Q.
      • Thoma C.R.
      • Izhar L.
      • Leng Y.
      • Guo A.
      • Chen Y.N.
      • Rush J.
      • Hsu P.W.
      • Yen H.C.
      • Elledge S.J.
      Global identification of modular Cullin-RING ligase substrates.
      ,
      • Wagner S.A.
      • Beli P.
      • Weinert B.T.
      • Nielsen M.L.
      • Cox J.
      • Mann M.
      • Choudhary C.
      A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles.
      ,
      • Udeshi N.D.
      • Mani D.R.
      • Eisenhaure T.
      • Mertins P.
      • Jaffe J.D.
      • Clauser K.R.
      • Hacohen N.
      • Carr S.A.
      Methods for quantification of in vivo changes in protein ubiquitination following proteasome and deubiquitinase inhibition.
      ,
      • Kim W.
      • Bennett E.J.
      • Huttlin E.L.
      • Guo A.
      • Li J.
      • Possemato A.
      • Sowa M.E.
      • Rad R.
      • Rush J.
      • Comb M.J.
      • Harper J.W.
      • Gygi S.P.
      Systematic and quantitative assessment of the ubiquitin-modified proteome.
      ). For example, it has been recently shown that detection of more than 20,000 unique ubiquitination sites is possible from the analysis of five different murine tissues (
      • Wagner S.A.
      • Beli P.
      • Weinert B.T.
      • Schölz C.
      • Kelstrup C.D.
      • Young C.
      • Nielsen M.L.
      • Olsen J.V.
      • Brakebusch C.
      • Choudhary C.
      Proteomic analyses reveal divergent ubiquitylation site patterns in murine tissues.
      ). However, as the authors indicate, only a few thousands sites are detected in any single analysis of an individual tissue sample (
      • Wagner S.A.
      • Beli P.
      • Weinert B.T.
      • Schölz C.
      • Kelstrup C.D.
      • Young C.
      • Nielsen M.L.
      • Olsen J.V.
      • Brakebusch C.
      • Choudhary C.
      Proteomic analyses reveal divergent ubiquitylation site patterns in murine tissues.
      ). It is recognized that there is need for further improvements in global ubiquitin technology to increase the depth-of-coverage attainable in quantitative proteomic experiments using moderate amounts of protein input (
      • Bustos D.
      • Bakalarski C.E.
      • Yang Y.
      • Peng J.
      • Kirkpatrick D.S.
      Characterizing ubiquitination sites by peptide-based immunoaffinity enrichment.
      ). Through systematic study and optimization of key pre-analytical variables in the preparation and use of the anti-K-ε-GG antibody as well as the proteomic workflow, we have now achieved, for the first time, routine quantification of ∼20,000 nonredundant K-ε-GG sites in a single SILAC triple encoded experiment starting with 5 mg of protein per SILAC channel. This represents a 10-fold improvement over our previously published method (
      • Udeshi N.D.
      • Mani D.R.
      • Eisenhaure T.
      • Mertins P.
      • Jaffe J.D.
      • Clauser K.R.
      • Hacohen N.
      • Carr S.A.
      Methods for quantification of in vivo changes in protein ubiquitination following proteasome and deubiquitinase inhibition.
      ).

      DISCUSSION

      In summary, we have described a number of critical enhancements to the K-ε-GG analysis pipeline that, taken together, have resulted in the most robust, sensitive, and practical method described to date for the analysis of endogenous ubiquitination sites from cellular lysates. The specific improvements we have introduced, including cross-linking of the antibody, optimization of the amount of antibody needed for efficient enrichment of K-ε-GG-peptides, as well as use of peptide separation methods that do not require desalting of sample prior to analysis by LC-MS/MS, all lend themselves to future automation of the technology for parallel processing of many samples simultaneously. We have shown, for the first time, that the depth-of-coverage obtainable from moderate amounts of starting material is on par with the degree of coverage obtained using highly optimized methods to detect protein phosphorylation sites (
      • Villén J.
      • Gygi S.P.
      The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry.
      ,
      • Ficarro S.B.
      • Adelmant G.
      • Tomar M.N.
      • Zhang Y.
      • Cheng V.J.
      • Marto J.A.
      Magnetic bead processor for rapid evaluation and optimization of parameters for phosphopeptide enrichment.
      ), moving the field closer to the goal of detecting and quantifying even low level ubiquitin sites from limiting amounts of protein input using the anti-K-ε-GG antibody (
      • Bustos D.
      • Bakalarski C.E.
      • Yang Y.
      • Peng J.
      • Kirkpatrick D.S.
      Characterizing ubiquitination sites by peptide-based immunoaffinity enrichment.
      ). We note that further increases in the number of ubiquitination sites detected and quantified in single experiments are possible, but they will likely require the use of more than 5 mg of input protein per label state combined with further decreases in sample complexity, for example by use of two-state versus three-state SILAC, or by peptide level fractionation into a larger number of fractions prior to enrichment. We anticipate that the techniques and refinements described in this work will not be limited in usefulness to only experiments using the anti-K-ε-GG antibody but will also be highly valuable in the future for optimization of other post-translational modification-specific antibodies.

      Acknowledgments

      We thank Jeffrey Silva and Charles Nogawe of Cell Signaling Technology for helpful discussions.

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