Analysis of Ubiquitinated Proteome by Quantitative Mass Spectrometry

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 893)

Abstract

Protein modification by ubiquitin (Ub) is one of the most common posttranslational events in eukaryotic cells. Ubiquitinated proteins are destined to various fates such as proteasomal degradation, protein trafficking, DNA repair, and immune response. In the last decade, vast improvements of mass spectrometry make it feasible to analyze the minute amount of ubiquitinated components in vivo. When combined with quantitative strategies, such as stable isotope labeling with amino acids in cell culture (SILAC), it is capable of profiling ubiquitinated proteome under different experimental conditions. Here, we describe a procedure to perform such a study, including differential protein labeling by the SILAC method, enrichment of ubiquitinated species, mass spectrometric analysis, and quality control to reduce false positives. The potential challenges and limitations of the procedure are also discussed.

Key words

Ubiquitin Proteomics Mass spectrometry SILAC 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgements

This work was partially supported by the National Institutes of Health grants (RR025822, and NS055077), and the American Cancer Society grant (RSG-09-181).

References

  1. 1.
    Semple CA (2003) The comparative proteomics of ubiquitination in mouse. Genome Res 13:1389–1394PubMedCrossRefGoogle Scholar
  2. 2.
    Schwartz AL, Ciechanover A (2009) Targeting proteins for destruction by the ubiquitin system: implications for human pathobiology. Annu Rev Pharmacol Toxicol 49:73–96PubMedCrossRefGoogle Scholar
  3. 3.
    Finley D (2009) Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu Rev Biochem 78:477–513PubMedCrossRefGoogle Scholar
  4. 4.
    Peng J, Schwartz D, Elias JE et al (2003) A proteomics approach to understanding protein ubiquitination. Nat Biotechnol 21:921–926PubMedCrossRefGoogle Scholar
  5. 5.
    Iwai K, Tokunaga F (2009) Linear polyubiquitination: a new regulator of NF-kappaB activation. EMBO Rep 10:706–713PubMedCrossRefGoogle Scholar
  6. 6.
    Pickart CM, Fushman D (2004) Polyubiquitin chains: polymeric protein signals. Curr Opin Chem Biol 8:610–616PubMedCrossRefGoogle Scholar
  7. 7.
    Mukhopadhyay D, Riezman H (2007) Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science 315:201–205PubMedCrossRefGoogle Scholar
  8. 8.
    Xu P, Duong DM, Seyfried NT et al (2009) Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137:133–145PubMedCrossRefGoogle Scholar
  9. 9.
    Jin L, Williamson A, Banerjee S et al (2008) Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex. Cell 133:653–665PubMedCrossRefGoogle Scholar
  10. 10.
    Bhoj VG, Chen ZJ (2009) Ubiquitylation in innate and adaptive immunity. Nature 458:430–437PubMedCrossRefGoogle Scholar
  11. 11.
    Hochstrasser M (2009) Origin and function of ubiquitin-like proteins. Nature 458:422–429PubMedCrossRefGoogle Scholar
  12. 12.
    Darwin KH (2009) Prokaryotic ubiquitin-like protein (Pup), proteasomes and pathogenesis. Nat Rev Microbiol 7:485–491PubMedCrossRefGoogle Scholar
  13. 13.
    Kirkpatrick DS, Denison C, Gygi SP (2005) Weighing in on ubiquitin: the expanding role of mass-spectrometry-based proteomics. Nat Cell Biol 7:750–757PubMedCrossRefGoogle Scholar
  14. 14.
    Xu P, Peng J (2006) Dissecting the ubiquitin pathway by mass spectrometry. Biochim Biophys Acta 1764:1940–1947PubMedCrossRefGoogle Scholar
  15. 15.
    Wang X, Guerrero C, Kaiser P et al (2007) Proteomics of proteasome complexes and ubiquitinated proteins. Expert Rev Proteomics 4:649–665PubMedCrossRefGoogle Scholar
  16. 16.
    Cravatt BF, Simon GM, Yates JR III (2007) The biological impact of mass-spectrometry-based proteomics. Nature 450:991–1000PubMedCrossRefGoogle Scholar
  17. 17.
    Gstaiger M, Aebersold R (2009) Applying mass spectrometry-based proteomics to genetics, genomics and network biology. Nat Rev Genet 10:617–627PubMedCrossRefGoogle Scholar
  18. 18.
    Choudhary C, Mann M (2010) Decoding signalling networks by mass spectrometry-based proteomics. Nat Rev Mol Cell Biol 11:427–439PubMedCrossRefGoogle Scholar
  19. 19.
    Peng J (2008) Evaluation of proteomic strategies for analyzing ubiquitinated proteins. BMB Rep 41:177–183PubMedCrossRefGoogle Scholar
  20. 20.
    Peng J, Cheng D (2005) Proteomic analysis of ubiquitin conjugates in yeast. Methods Enzymol 399:367–381PubMedCrossRefGoogle Scholar
  21. 21.
    Tagwerker C, Flick K, Cui M et al (2006) 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. Mol Cell Proteomics 5:737–748PubMedGoogle Scholar
  22. 22.
    Meierhofer D, Wang X, Huang L et al (2008) Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry. J Proteome Res 7:4566–4576PubMedCrossRefGoogle Scholar
  23. 23.
    Matsumoto M, Hatakeyama S, Oyamada K et al (2005) Large-scale analysis of the human ubiquitin-related proteome. Proteomics 5:4145–4151PubMedCrossRefGoogle Scholar
  24. 24.
    Vasilescu J, Smith JC, Ethier M et al (2005) Proteomic analysis of ubiquitinated proteins from human MCF-7 breast cancer cells by immunoaffinity purification and mass spectrometry. J Proteome Res 4:2192–2200PubMedCrossRefGoogle Scholar
  25. 25.
    Newton K, Matsumoto ML, Wertz IE et al (2008) Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies. Cell 134:668–678PubMedCrossRefGoogle Scholar
  26. 26.
    Layfield R, Tooth D, Landon M et al (2001) Purification of poly-ubiquitinated proteins by S5a-affinity chromatography. Proteomics 1:773–777PubMedCrossRefGoogle Scholar
  27. 27.
    Weekes J, Morrison K, Mullen A et al (2003) Hyperubiquitination of proteins in dilated cardiomyopathy. Proteomics 3:208–216PubMedCrossRefGoogle Scholar
  28. 28.
    Maor R, Jones A, Nuhse TS et al (2007) Multidimensional protein identification technology (MudPIT) analysis of ubiquitinated proteins in plants. Mol Cell Proteomics 6:601–610PubMedCrossRefGoogle Scholar
  29. 29.
    Bennett EJ, Shaler TA, Woodman B et al (2007) Global changes to the ubiquitin system in Huntington’s disease. Nature 448:704–708PubMedCrossRefGoogle Scholar
  30. 30.
    Hjerpe R, Aillet F, Lopitz-Otsoa F et al (2009) Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. EMBO Rep 10:1250–1258PubMedCrossRefGoogle Scholar
  31. 31.
    Peng J, Gygi SP (2001) Proteomics: the move to mixtures. J Mass Spectrom 36:1083–1091PubMedCrossRefGoogle Scholar
  32. 32.
    Marotti LA Jr, Newitt R, Wang Y et al (2002) Direct identification of a G protein ubiquitination site by mass spectrometry. Biochemistry 41:5067–5074PubMedCrossRefGoogle Scholar
  33. 33.
    Seyfried NT, Xu P, Duong DM et al (2008) Systematic approach for validating the ubiquitinated proteome. Anal Chem 80:4161–4169PubMedCrossRefGoogle Scholar
  34. 34.
    Mayor T, Graumann J, Bryan J et al (2007) Quantitative profiling of ubiquitylated proteins reveals proteasome substrates and the substrate repertoire influenced by the Rpn10 receptor pathway. Mol Cell Proteomics 6:1885–1895PubMedCrossRefGoogle Scholar
  35. 35.
    Xu P, Duong DM, Peng J (2009) Systematical optimization of reverse-phase chromatography for shotgun proteomics. J Proteome Res 8:3944–3950PubMedCrossRefGoogle Scholar
  36. 36.
    Eng J, McCormack AL, Yates JR 3rd (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5:976–989CrossRefGoogle Scholar
  37. 37.
    Peng J, Elias JE, Thoreen CC et al (2003) Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. J Proteome Res 2:43–50PubMedCrossRefGoogle Scholar
  38. 38.
    Elias JE, Gygi SP (2007) Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat Methods 4:207–214PubMedCrossRefGoogle Scholar
  39. 39.
    Seyfried NT, Gozal YM, Dammer EB et al (2010) Multiplex SILAC analysis of a cellular TDP-43 proteinopathy model reveals protein inclusions associated with SUMOylation and diverse polyubiquitin chains. Mol Cell Proteomics 9:705–718PubMedCrossRefGoogle Scholar
  40. 40.
    Shevchenko A, Wilm M, Vorm O et al (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68:850–858PubMedCrossRefGoogle Scholar
  41. 41.
    de Godoy LM, Olsen JV, Cox J et al (2008) Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast. Nature 455:1251–1254PubMedCrossRefGoogle Scholar
  42. 42.
    Sury MD, Chen JX, Selbach M (2010) The SILAC fly allows for accurate protein quantification in vivo. Mol Cell Proteomics 9(10):2173–2178PubMedCrossRefGoogle Scholar
  43. 43.
    Kruger M, Moser M, Ussar S et al (2008) SILAC mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function. Cell 134:353–364PubMedCrossRefGoogle Scholar
  44. 44.
    Nielsen ML, Vermeulen M, Bonaldi T et al (2008) Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nat Methods 5:459–460PubMedCrossRefGoogle Scholar
  45. 45.
    Xu P, Cheng D, Duong DM et al (2006) A proteomic strategy for quantifying polyubiquitin chain topologies. Israel J Chem 46:171–182CrossRefGoogle Scholar
  46. 46.
    Golebiowski F, Matic I, Tatham MH et al (2009) System-wide changes to SUMO modifications in response to heat shock. Sci Signal 2:ra24Google Scholar
  47. 47.
    Xu G, Paige JS, Jaffrey SR (2010) Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nat Biotechnol 28:868–873PubMedCrossRefGoogle Scholar
  48. 48.
    Ross PL, Huang YN, Marchese JN et al (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3:1154–1169PubMedCrossRefGoogle Scholar
  49. 49.
    Kirkpatrick DS, Hathaway NA, Hanna J et al (2006) Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology. Nat Cell Biol 8:700–710PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Structural BiologySt. Jude Children’s Research HospitalMemphisUSA
  2. 2.Department Developmental NeurobiologySt. Jude Children’s Research HospitalMemphisUSA

Personalised recommendations