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Quantitative Profiling of Ubiquitylated Proteins Reveals Proteasome Substrates and the Substrate Repertoire Influenced by the Rpn10 Receptor Pathway*

  • Thibault Mayor
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, UBC Centre for Proteomics, University of British Columbia, 301-2185 East Mall, Vancouver, British Columbia V6T 1Z4, Canada. Tel.: 604-822-5144; Fax: 604-822-2114;
    Affiliations
    Howard Hughes Medical Institute, Division of Biology, California Institute of Technology, Pasadena, California 91125

    Department of Biochemistry and Molecular Biology, UBC Centre for Proteomics, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
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  • Johannes Graumann
    Affiliations
    Howard Hughes Medical Institute, Division of Biology, California Institute of Technology, Pasadena, California 91125
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  • Jennifer Bryan
    Footnotes
    Affiliations
    Department of Statistics, Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
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  • Michael J. MacCoss
    Footnotes
    Affiliations
    Department of Genome Sciences, Health Sciences Center, University of Washington, Seattle, Washington 98195
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  • Raymond J. Deshaies
    Footnotes
    Affiliations
    Howard Hughes Medical Institute, Division of Biology, California Institute of Technology, Pasadena, California 91125
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  • Author Footnotes
    * This work was supported in part by the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
    ** Supported by Natural Sciences and Engineering Research Council (Canada) and the Michael Smith Foundation for Health Research.
    §§ Supported by National Institutes of Health Grant P41-RR011823.
    ¶¶ An Investigator of the Howard Hughes Medical Institute.
Open AccessPublished:July 20, 2007DOI:https://doi.org/10.1074/mcp.M700264-MCP200
      The ubiquitin proteasome system (UPS) comprises hundreds of different conjugation/deconjugation enzymes and multiple receptors that recognize ubiquitylated proteins. A formidable challenge to deciphering the biology of ubiquitin is to map the networks of substrates and ligands for components of the UPS. Several different receptors guide ubiquitylated substrates to the proteasome, and neither the basis for specificity nor the relative contribution of each pathway is known. To address how broad of a role the ubiquitin receptor Rpn10 (S5a) plays in turnover of proteasome substrates, we implemented a method to perform quantitative analysis of ubiquitin conjugates affinity-purified from experimentally perturbed and reference cultures of Saccharomyces cerevisiae that were differentially labeled with 14N and 15N isotopes. Shotgun mass spectrometry coupled with relative quantification using metabolic labeling and statistical analysis based on q values revealed ubiquitylated proteins that increased or decreased in level in response to a particular treatment. We first identified over 225 candidate UPS substrates that accumulated as ubiquitin conjugates upon proteasome inhibition. To determine which of these proteins were influenced by Rpn10, we evaluated the ubiquitin conjugate proteomes in cells lacking either the entire Rpn10 (rpn10Δ) (or only its UIM (ubiquitin-interacting motif) polyubiquitin-binding domain (uimΔ)). Twenty-seven percent of the UPS substrates accumulated as ubiquitylated species in rpn10Δ cells, whereas only one-fifth as many accumulated in uimΔ cells. These findings underscore a broad role for Rpn10 in turnover of ubiquitylated substrates but a relatively modest role for its ubiquitin-binding UIM domain. This approach illustrates the feasibility of systems-level quantitative analysis to map enzyme-substrate networks in the UPS.
      The classical function of ubiquitylation is to direct substrates for proteolysis via the ubiquitin proteasome system (UPS).
      The abbreviations used are: UPS, ubiquitin proteasome system; VWA, von Willebrand A; UIM, ubiquitin-interacting motif; FDR, false discovery rate; TAP, tandem affinity purification; MudPIT, multidimensional protein identification technology; WT, wild-type; DUB, deubiquitylating.
      1The abbreviations used are: UPS, ubiquitin proteasome system; VWA, von Willebrand A; UIM, ubiquitin-interacting motif; FDR, false discovery rate; TAP, tandem affinity purification; MudPIT, multidimensional protein identification technology; WT, wild-type; DUB, deubiquitylating.
      Recognition of proteasome substrates is specifically mediated by several receptor proteins (
      • Madura K.
      Rad23 and Rpn10: perennial wallflowers join the melee.
      ). In yeast, there are at least five potential receptors (Ddi1, Dsk2, Rad23, Rpn10, and Rpt5) plus a set of Cdc48-based complexes, including the Cdc48-Npl4-Ufd1 heterotrimer, that may possess receptor function (
      • Elsasser S.
      • Chandler-Militello D.
      • Muller B.
      • Hanna J.
      • Finley D.
      Rad23 and Rpn10 serve as alternative ubiquitin receptors for the proteasome.
      ,
      • Ivantsiv Y.
      • Kaplun L.
      • Tzirkin-Goldin R.
      • Shabek N.
      • Raveh D.
      Unique role for the UbL-UbA protein Ddi1 in turnover of SCFUfo1 complexes.
      ,
      • Medicherla B.
      • Kostova Z.
      • Schaefer A.
      • Wolf D.H.
      A genomic screen identifies Dsk2p and Rad23p as essential components of ER-associated degradation.
      ,
      • Verma R.
      • Oania R.
      • Graumann J.
      • Deshaies R.J.
      Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system.
      ,
      • Lam Y.A.
      • Lawson T.G.
      • Velayutham M.
      • Zweier J.L.
      • Pickart C.M.
      A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal.
      ,
      • Hartmann-Petersen R.
      • Wallace M.
      • Hofmann K.
      • Koch G.
      • Johnsen A.H.
      • Hendil K.B.
      • Gordon C.
      The Ubx2 and Ubx3 cofactors direct Cdc48 activity to proteolytic and nonproteolytic ubiquitin-dependent processes.
      ). This diversity of postubiquitylation targeting pathways is mystifying. Currently it is not known which subset of proteasome substrates is targeted by a given receptor or what features govern the allocation of substrates to a particular receptor pathway.
      The yeast Rpn10 protein is a stoichiometric component of the 26 S proteasome and was the first protein found to bind polyubiquitin chains (
      • Deveraux Q.
      • Ustrell V.
      • Pickart C.
      • Rechsteiner M.
      A 26 S protease subunit that binds ubiquitin conjugates.
      ). Its amino-terminal domain consists of a conserved von Willebrand A (VWA) motif that docks Rpn10 to the proteasome. Recruitment of ubiquitin chains to Rpn10 is mediated by the 20-amino acid ubiquitin-interacting motif (UIM) domain located near its carboxyl terminus (
      • Hofmann K.
      • Falquet L.
      A ubiquitin-interacting motif conserved in components of the proteasomal and lysosomal protein degradation systems.
      ). S5a protein, the human Rpn10 ortholog, contains a second UIM domain that is thought to mediate the recruitment of other receptor proteins (
      • Hiyama H.
      • Yokoi M.
      • Masutani C.
      • Sugasawa K.
      • Maekawa T.
      • Tanaka K.
      • Hoeijmakers J.H.
      • Hanaoka F.
      Interaction of hHR23 with S5a. The ubiquitin-like domain of hHR23 mediates interaction with S5a subunit of 26 S proteasome.
      ). The general impact of Rpn10 on the turnover of proteasome substrates is not known. Given that budding yeast rpn10Δ mutants are viable (
      • van Nocker S.
      • Sadis S.
      • Rubin D.M.
      • Glickman M.
      • Fu H.
      • Coux O.
      • Wefes I.
      • Finley D.
      • Vierstra R.D.
      The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover.
      ,
      • Fu H.
      • Sadis S.
      • Rubin D.M.
      • Glickman M.
      • van Nocker S.
      • Finley D.
      • Vierstra R.D.
      Multiubiquitin chain binding and protein degradation are mediated by distinct domains within the 26 S proteasome subunit Mcb1.
      ), Rpn10 may be required for the turnover of only a small subset of ubiquitylated proteins, or Rpn10 may target a large number of substrates that, in its absence, are targeted by other proteasomal receptors (e.g. Rad23 or Dsk2). Even less well understood is the contribution of the two domains of Rpn10 to substrate turnover. Complete deletion of RPN10 (i.e. rpn10Δ) stabilizes the cell cycle regulator Sic1 and the transcription factor Gcn4. Paradoxically removal of the UIM domain by itself (i.e. uimΔ) has no discernable effect on either of these substrates (
      • Verma R.
      • Oania R.
      • Graumann J.
      • Deshaies R.J.
      Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system.
      )
      J. R. Lipford, personal communication.
      2J. R. Lipford, personal communication.
      suggesting that Rpn10 function may rely solely on an uncharacterized biochemical activity associated with its VWA domain.
      To understand fully the biological roles of protein ubiquitylation and the functions of individual components of the UPS such as Rpn10, it will be necessary to identify UPS substrates on a proteome-wide scale. Several studies have started to address this challenge using mass spectrometry to analyze the ubiquitin proteome (
      • Kirkpatrick D.S.
      • Weldon S.F.
      • Tsaprailis G.
      • Liebler D.C.
      • Gandolfi A.J.
      Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin.
      ,
      • Matsumoto M.
      • Hatakeyama S.
      • Oyamada K.
      • Oda Y.
      • Nishimura T.
      • Nakayama K.I.
      Large-scale analysis of the human ubiquitin-related proteome.
      ,
      • Mayor T.
      • Lipford J.R.
      • Graumann J.
      • Smith G.T.
      • Deshaies R.J.
      Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets.
      ,
      • Peng J.
      • Schwartz D.
      • Elias J.E.
      • Thoreen C.C.
      • Cheng D.
      • Marsischky G.
      • Roelofs J.
      • Finley D.
      • Gygi S.P.
      A proteomics approach to understanding protein ubiquitination.
      ,
      • Tagwerker C.
      • Flick K.
      • Cui M.
      • Guerrero C.
      • Dou Y.
      • Auer B.
      • Baldi P.
      • Huang L.
      • Kaiser P.A.
      A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivo cross-linking.
      ,
      • Hitchcock A.L.
      • Auld K.
      • Gygi S.P.
      • Silver P.A.
      A subset of membrane-associated proteins is ubiquitinated in response to mutations in the endoplasmic reticulum degradation machinery.
      ). Although these seminal studies illustrate that shotgun mass spectrometry is a powerful tool that can provide a systems-level view of the ubiquitin proteome, it is clear that application of this technology to the ubiquitin system remains in an embryonic state. For example, no proteomics study has yet succeeded in identifying even one of the 11 yeast G1 and mitotic cyclins that are well known substrates of the UPS. Indeed many ubiquitin conjugates identified in proteomics experiments might be stably accumulating species that are not substrates of the UPS. To obtain more focused information from shotgun mass spectrometry experiments, we and others have previously applied subtractive approaches to identify conjugates that accumulate in rpn10Δ (
      • Mayor T.
      • Lipford J.R.
      • Graumann J.
      • Smith G.T.
      • Deshaies R.J.
      Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets.
      ) and in npl4ts but not ubc7Δ mutants (
      • Hitchcock A.L.
      • Auld K.
      • Gygi S.P.
      • Silver P.A.
      A subset of membrane-associated proteins is ubiquitinated in response to mutations in the endoplasmic reticulum degradation machinery.
      ). Although this strategy allowed the identification of several ubiquitylated proteins, by its nature the subtractive approach excludes substrates whose accumulation is only partially dependent upon a given factor. This is a major concern given the redundancy of many UPS pathways (
      • Verma R.
      • Oania R.
      • Graumann J.
      • Deshaies R.J.
      Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system.
      ,
      • Chi Y.
      • Huddleston M.J.
      • Zhang X.
      • Young R.A.
      • Annan R.S.
      • Carr S.A.
      • Deshaies R.J.
      Negative regulation of Gcn4 and Msn2 transcription factors by Srb10 cyclin-dependent kinase.
      ). No fewer than six ubiquitin ligases (Mdm2, Pirh2, p300, PARC, Cul7, and Cop1) have been implicated in p53 regulation (
      • Andrews P.
      • He Y.J.
      • Xiong Y.
      Cytoplasmic localized ubiquitin ligase cullin 7 binds to p53 and promotes cell growth by antagonizing p53 function.
      ,
      • Brooks C.L.
      • Gu W.
      p53 ubiquitination: Mdm2 and beyond.
      ,
      • Grossman S.R.
      • Deato M.E.
      • Brignone C.
      • Chan H.M.
      • Kung A.L.
      • Tagami H.
      • Nakatani Y.
      • Livingston D.M.
      Polyubiquitination of p53 by a ubiquitin ligase activity of p300.
      ), and at least three different ubiquitin chain receptors contribute to turnover of ubiquitylated Sic1 (
      • Verma R.
      • Oania R.
      • Graumann J.
      • Deshaies R.J.
      Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system.
      ). Clearly a method that allows for more subtle quantitative comparisons is needed.
      In this study, we adapted stable isotope labeling techniques that have been used previously to address a variety of biological problems to perform relative quantitative analysis of polyubiquitylated proteins in two distinct cell cultures. By applying a statistical approach based on p and q values, we were able to identify ubiquitylated proteins whose levels are altered in response to a specific perturbation (chemical or genetic). After validating the approach, we used this method to identify putative substrates of the proteasome and to determine the contribution of the Rpn10 proteasome receptor pathway in the targeting of UPS substrates. We further dissected the function of Rpn10 by assessing the role of its UIM domain.

      DISCUSSION

      We performed quantitative analysis using 15N metabolic labeling to measure variations in levels of ubiquitin conjugates after chemical and genetic perturbations. In a first series of experiments, we were able to specifically identify UPS substrates using the proteasome inhibitor MG132. We then extended our analysis to identify ubiquitylated proteins that are affected by the Rpn10 pathway. This enabled the identification of several ubiquitylated substrates whose metabolism is influenced by the UIM domain of Rpn10. Finally we compared the different analyses to gauge the relative impact of deleting sequences encoding either the entire RPN10 or only its UIM domain on the UPS proteome.

      The Rpn10 Pathway—

      By comparing datasets for ubiquitin conjugates that accumulate when the proteasome is inhibited with MG132 (putative UPS substrates) and those that accumulate in rpn10Δ cells, we estimated that Rpn10 influenced the steady-state level of ubiquitin conjugates for up to ∼27% of all UPS substrates (Fig. 5). The simplest interpretation of this result is that Rpn10 contributes to the turnover of a significant number of ubiquitylated proteins. However, we cannot exclude the possibility that in some cases the role of Rpn10 in turnover is indirect or that the increase in conjugates is due to increased ubiquitylation or decreased deubiquitylation. Interestingly there were also several proteins that were significantly de-enriched in rpn10Δ cells suggesting that Rpn10 function might be broader than suspected. Given that rpn10Δ cells exhibit mild phenotypes (
      • Fu H.
      • Sadis S.
      • Rubin D.M.
      • Glickman M.
      • van Nocker S.
      • Finley D.
      • Vierstra R.D.
      Multiubiquitin chain binding and protein degradation are mediated by distinct domains within the 26 S proteasome subunit Mcb1.
      ) this important impact of rpn10Δ in our experiments is somewhat surprising. Presumably the “Rpn10-dependent” candidate substrates reported here do not rely exclusively on Rpn10 for delivery to the proteasome. Instead it is more likely that substrates that use Rpn10 can also use other receptor pathways, albeit with reduced overall efficiency, when Rpn10 is absent as is the case for Sic1 (
      • Verma R.
      • Oania R.
      • Graumann J.
      • Deshaies R.J.
      Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system.
      ).
      The role of the UIM domain of Rpn10 in the UPS has been perplexing. Although it was the first polyubiquitin binding domain to be identified, it remained unclear whether it plays any role in proteolysis in wild-type cells. The Rpn10-dependent substrates Sic1 and Gcn4 are unaffected by the UIM deletion unless it is combined with deletions of other receptors such as RAD23 (
      • Verma R.
      • Oania R.
      • Graumann J.
      • Deshaies R.J.
      Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system.
      ).
      J. R. Lipford, personal communication.
      In this analysis we identified several ubiquitylated substrates that accumulated in uimΔ cells (Supplemental Table 8). To our knowledge, these represent the first physiological UPS substrates that have been shown to be affected by loss of the UIM domain by itself. This shows that there is a dual role for Rpn10 function at the proteome-wide level (Fig. 5). Those identified proteins can now be used as “indicator proteins” to unravel the physiological role of the UIM domain. The notion of using mass spectrometry to identify an “indicator protein” should be readily applicable to other poorly understood components of the UPS.
      Of the proteins accumulating as ubiquitylated species upon RPN10 deletion, only one-fifth accumulated upon the selective deletion of the UIM domain. This is consistent with the observation that rpn10Δ cells have a more severe growth defect than uimΔ cells (
      • Fu H.
      • Sadis S.
      • Rubin D.M.
      • Glickman M.
      • van Nocker S.
      • Finley D.
      • Vierstra R.D.
      Multiubiquitin chain binding and protein degradation are mediated by distinct domains within the 26 S proteasome subunit Mcb1.
      ). The substrates that accumulated in rpn10Δ but not uimΔ cells presumably are dependent upon the VWA domain. The VWA domain enhances the degradation of ubiquitylated Sic1 docked to the proteasome by the Rad23 receptor, but the biochemical function of the VWA domain remains unknown. Interestingly ribosome and DNA-associated proteins were found to be significantly enriched in the Rpn10 analysis but not in the UIM analysis (data not shown). These proteins are part of large and tight complexes. It is possible that the VWA might participate in the structural conformation of the 19 S subunit of the proteasome, favoring a larger entry site for the substrate or better alignment with other proteasomal functions (e.g. Rpn11-DUB or the chaperone activities of Rpt).

      The UPS Proteome—

      Our analysis revealed a large number of ubiquitylated proteins that accumulated upon inhibition of the proteasome, including relatively low abundance cell cycle control proteins whose activity is known to be regulated by proteolysis (G1 cyclins Cln1, Cln2, and Pcl1; B-type cyclin Clb2; and Cdk inhibitors Sic1 and Far1). Interestingly we also found and confirmed that abundant, presumably stable proteins (ribosomal protein Rps8A, enolase, and phosphoglycerate kinase) accumulated as ubiquitylated species upon inhibition of the proteasome with MG132 (Fig. 2B). It is possible that these proteins succumbed to quality control mechanisms that eliminate improperly translated, misfolded, or damaged proteins (
      • McClellan A.J.
      • Tam S.
      • Kaganovich D.
      • Frydman J.
      Protein quality control: chaperones culling corrupt conformations.
      ,
      • Princiotta M.F.
      • Finzi D.
      • Qian S.B.
      • Gibbs J.
      • Schuchmann S.
      • Buttgereit F.
      • Bennink J.R.
      • Yewdell J.W.
      Quantitating protein synthesis, degradation, and endogenous antigen processing.
      ). If so, the methods reported here may be useful for studying on a proteome-wide scale the chaperones involved in protein folding and assembly. An important feature of our method is that we use consecutive affinity purification steps to focus our analysis on proteasome substrates and bias against contaminating proteins. The effectiveness of this strategy is underscored by the fact that >22% of our candidate UPS substrates are present at <1000 molecules/cell, whereas only 6.8% of proteins identified in a single MudPIT analysis of crude extract are of equivalent abundance (
      • Liu H.
      • Sadygov R.G.
      • Yates III, J.R.
      A model for random sampling and estimation of relative protein abundance in shotgun proteomics.
      ). The positive attributes of this method suggest that it should be generally useful for identifying targets of specific ubiquitin ligases and deubiquitylating enzymes.

      General Issues in Quantitative Profiling of UPS Substrates—

      The accumulation of a particular substrate in any given experiment was likely guided by several factors, including the amount of co-accumulating substrates, the relative activity of individual ubiquitin ligases and DUB enzymes that act upon the substrate, the relative ability of different ubiquitylation pathways to incorporate His6-ubiquitin or compete for free ubiquitin, and the degradation rate of the ubiquitylated protein. Because of these factors, we believe there is little value in investing great significance in individual ratios when drawing conclusions in a proteomics manner. Thus, we limited our analysis to classifying proteins as being enriched or not.
      In many cases the enrichment values for UPS substrates were lower than the threshold of 4 (or 2 when expressed in log2 scale) often applied in microarray analyses of mRNA expression (
      • Hoheisel J.D.
      Microarray technology: beyond transcript profiling and genotype analysis.
      ). In our experiments, the total enrichment for ubiquitin recovered from MG132-treated compared with untreated cells averaged only ∼1.9-fold (in Fig. 1). Thus, one would expect the average ubiquitylated substrate to accumulate 2–3-fold. This value agrees with immunoblot analysis of ubiquitin conjugates in total cell extracts. Although we could have increased this value by longer treatment with MG132, excessive accumulation of conjugates runs the risk of depleting cellular ubiquitin, thereby causing its redistribution to different proteins (
      • Mimnaugh E.G.
      • Chen H.Y.
      • Davie J.R.
      • Celis J.E.
      • Neckers L.
      Rapid deubiquitination of nucleosomal histones in human tumor cells caused by proteasome inhibitors and stress response inducers: effects on replication, transcription, translation, and the cellular stress response.
      ,
      • Dantuma N.P.
      • Groothuis T.A.
      • Salomons F.A.
      • Neefjes J.
      A dynamic ubiquitin equilibrium couples proteasomal activity to chromatin remodeling.
      ). Remarkably despite the limited extent of accumulation of total ubiquitin conjugates, large variations in accumulation of specific conjugates were seen.
      We used a statistical analysis based on q values to identify proteins for which the ubiquitylation level was altered. The reference experiment, in which two biologically equivalent pools were compared, provided crucial information regarding the typical variation arising from simple biological variability and experimental noise. Protein-specific p values provide a statistical measure of the inconsistency between the observed log ratio and a null hypothesis of no enrichment or depletion. We further modified our p values prior to forming lists to address the perennial problem in “omic” analyses, namely the large scale multiple testing problem. A simple filter for p values only allows a selection based on the error rate (proportion of unaffected ORFs that are considered enriched, i.e. false negative), whereas q values allow the setting of a threshold FDR (based on false positive rate). By setting a q value threshold of 0.05 (or 0.15 in the UIM analysis), the lists of putative proteome substrates have, on average, a false positive proportion smaller than 5% (or 15%). Note that in all experiments the error rates were below or close to 10%. In addition to the absolute meaning of the q value cutoff in terms of the FDR, our choice of cutoff was guided by a desire to recover a high proportion (which we estimated to be close to 80% in most of our analyses) of the ORFs that responded to a chemical (MG132) or genetic (rpn10Δ, uimΔ) perturbation of the UPS. Manual validation of a subset of proteins from the MG132 and uimΔ analyses confirmed the efficacy of the approach. To our best knowledge, this is the first analysis that combines the null distribution of a reference experiment and q value test to identify responsive or affected proteins in quantitative mass spectrometry analysis. This method offers considerable promise that could be broadly applied to other proteomics studies.

      Acknowledgments

      We thank Brian Williams for help in the list analysis and Barbara J. Wold, Rati Verma, Robert Riley, Sonja Hess, Allan D. Drummond, and Kenneth McCue for discussion. We thank all current and past members of the Deshaies laboratory for help, in particular Geoff T. Smith for dedicated technical assistance. T. M. also thanks Gary Kleiger for discussions, encouragement, and shared coffee breaks.

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