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Profiling Lysine Ubiquitination by Selective Enrichment of Ubiquitin Remnant-Containing Peptides

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Exocytosis and Endocytosis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1174))

Abstract

Protein ubiquitination plays critical roles in many biological processes. However, functional studies of protein ubiquitination in eukaryotic cells are limited by the ability to identify protein ubiquitination sites. Unbiased high-throughput screening methods are necessary to discover novel ubiquitination sites that play important roles in cellular regulation. Here, we describe an immunopurification approach that enriches ubiquitin remnant-containing peptides to facilitate downstream mass spectrometry (MS) identification of lysine ubiquitination sites. This approach can be utilized to identify ubiquitination sites from proteins in a complex mixture.

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References

  1. Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67:425–479

    Article  CAS  PubMed  Google Scholar 

  2. Peng J, Schwartz D, Elias JE et al (2003) A proteomics approach to understanding protein ubiquitination. Nat Biotechnol 21: 921–926

    Article  CAS  PubMed  Google Scholar 

  3. Xu G, Paige JS, Jaffrey SR (2010) Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nat Biotechnol 28:868–873

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Beers EP, Callis J (1993) Utility of polyhistidine-tagged ubiquitin in the purification of ubiquitin-protein conjugates and as an affinity ligand for the purification of ubiquitin-specific hydrolases. J Biol Chem 268:21645–21649

    CAS  PubMed  Google Scholar 

  5. Cooper HJ, Heath JK, Jaffray E, Hay RT, Lam TT, Marshall AG (2004) Identification of sites of ubiquitination in proteins: a fourier transform ion cyclotron resonance mass spectrometry approach. Anal Chem 76:6982–6988

    Article  CAS  PubMed  Google Scholar 

  6. Jeon HB, Choi ES, Yoon JH et al (2007) A proteomics approach to identify the ubiquitinated proteins in mouse heart. Biochem Biophys Res Commun 357:731–736

    Article  CAS  PubMed  Google Scholar 

  7. Layfield R, Tooth D, Landon M, Dawson S, Mayer J, Alban A (2001) Purification of poly-ubiquitinated proteins by S5a-affinity chromatography. Proteomics 1:773–777

    Article  CAS  PubMed  Google Scholar 

  8. Matsumoto M, Hatakeyama S, Oyamada K, Oda Y, Nishimura T, Nakayama KI (2005) Large-scale analysis of the human ubiquitin-related proteome. Proteomics 5:4145–4151

    Article  CAS  PubMed  Google Scholar 

  9. Tagwerker C, Flick K, Cui M, Guerrero C, Dou Y, Auer B 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 5:737–748

    Article  CAS  PubMed  Google Scholar 

  10. Srikumar T, Jeram SM, Lam H, Raught B (2010) A ubiquitin and ubiquitin-like protein spectral library. Proteomics 10:337–342

    Article  CAS  PubMed  Google Scholar 

  11. Tomlinson E, Palaniyappan N, Tooth D, Layfield R (2007) Methods for the purification of ubiquitinated proteins. Proteomics 7: 1016–1022

    Article  CAS  PubMed  Google Scholar 

  12. Xu P, Peng J (2006) Dissecting the ubiquitin pathway by mass spectrometry. Biochim Biophys Acta 1764:1940–1947

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Vasilescu J, Smith JC, Ethier M, Figeys D (2005) Proteomic analysis of ubiquitinated proteins from human MCF-7 breast cancer cells by immunoaffinity purification and mass spectrometry. J Proteome Res 4:2192–2200

    Article  CAS  PubMed  Google Scholar 

  14. Radivojac P, Vacic V, Haynes C, Cocklin RR, Mohan A, Heyen JW et al (2010) Identification, analysis, and prediction of protein ubiquitination sites. Proteins 78:365–380

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Maor R, Jones A, Nuhse TS, Studholme DJ, Peck SC, Shirasu K (2007) Multidimensional protein identification technology (MudPIT) analysis of ubiquitinated proteins in plants. Mol Cell Proteomics 6:601–610

    Article  CAS  PubMed  Google Scholar 

  16. Smale ST (2010) Calcium phosphate transfection of 3T3 fibroblasts. Cold Spring Harb Protoc. doi:10.1101/pdb.prot5372

    Google Scholar 

  17. Kaiser P, Wohlschlegel J (2005) Identification of ubiquitination sites and determination of ubiquitin-chain architectures by mass spectrometry. Methods Enzymol 399:266–277

    Article  CAS  PubMed  Google Scholar 

  18. Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860

    Article  CAS  PubMed  Google Scholar 

  19. Nielsen ML, Vermeulen M, Bonaldi T, Cox J, Moroder L, Mann M (2008) Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nat Methods 5:459–460

    Article  CAS  PubMed  Google Scholar 

  20. Link AJ, LaBaer J (2011) Solution protein digest. Cold Spring Harb Protoc. doi:10.1101/pdb.prot5569

    Google Scholar 

  21. Parker CE, Warren MR, Mocanu V, Greer SF, Borchers CH (2008) Mass spectrometric determination of protein ubiquitination. Methods Mol Biol 446:109–130

    Article  CAS  PubMed  Google Scholar 

  22. Harlow E, Lane D (2006) Immunoaffinity purification: coupling antibodies to protein A or G bead columns. Cold Spring Harb Protoc. doi: 10.1101/pdb.prot4303

  23. Mattson G, Conklin E, Desai S, Nielander G, Savage MD, Morgensen S (1993) A practical approach to crosslinking. Mol Biol Rep 17: 167–183

    Article  CAS  PubMed  Google Scholar 

  24. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20: 3551–3567

    Article  CAS  PubMed  Google Scholar 

  25. Craig R, Beavis RC (2004) TANDEM: matching proteins with tandem mass spectra. Bioinformatics 20:1466–1467

    Article  CAS  PubMed  Google Scholar 

  26. Geer LY, Markey SP, Kowalak JA, Wagner L, Xu M, Maynard DM et al (2004) Open mass spectrometry search algorithm. J Proteome Res 3:958–964

    Article  CAS  PubMed  Google Scholar 

  27. MacCoss MJ, Wu CC, Yates JR 3rd (2002) Probability-based validation of protein identifications using a modified SEQUEST algorithm. Anal Chem 74:5593–5599

    Article  CAS  PubMed  Google Scholar 

  28. Palagi PM, Hernandez P, Walther D, Appel RD (2006) Proteome informatics I: bioinformatics tools for processing experimental data. Proteomics 6:5435–5444

    Article  CAS  PubMed  Google Scholar 

  29. Boeckmann B, Bairoch A, Apweiler R, Blatter MC, Estreicher A, Gasteiger E et al (2003) The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003. Nucleic Acids Res 31:365–370

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Pruitt KD, Tatusova T, Maglott DR (2007) NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res 35:D61–D65

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kersey PJ, Duarte J, Williams A, Karavidopoulou Y, Birney E, Apweiler R (2004) The International Protein Index: an integrated database for proteomics experiments. Proteomics 4:1985–1988

    Article  CAS  PubMed  Google Scholar 

  32. Xu G, Jaffrey SR (2013) Proteomic identification of protein ubiquitination events. Biotechnol Genet Eng Rev 29:73–109

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Seo J, Jeong J, Kim YM, Hwang N, Paek E, Lee KJ (2008) Strategy for comprehensive identification of post-translational modifications in cellular proteins, including low abundant modifications: application to glyceraldehyde-3-phosphate dehydrogenase. J Proteome Res 7:587–602

    Article  CAS  PubMed  Google Scholar 

  34. Xu H, Freitas MA (2009) Automated diagnosis of LC-MS/MS performance. Bioinformatics 25: 1341–1343

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The work was supported by the National Natural Science Foundation of China (Grant 31270874), Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases (BM2013003), a project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions (GX), NIH-NIMH (MH086128) (SRJ), Boehringer Ingelheim Fonds predoctoral fellowship (AD).

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Authors and Affiliations

  1. Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China

    Guoqiang Xu

  2. Department of Pharmacology, Weill Medical College, Cornell University, 1300 York Avenue, Room LC-523, 70, New York, 10065, NY, USA

    Alessia Deglincerti, Jeremy S. Paige & Samie R. Jaffrey

Authors
  1. Guoqiang Xu
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  2. Alessia Deglincerti
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  3. Jeremy S. Paige
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  4. Samie R. Jaffrey
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Corresponding author

Correspondence to Samie R. Jaffrey .

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Editors and Affiliations

  1. Department of Human and Molecular Genetics, VCU Institute of Molecular Medicine, Virginia Commonwealth University, Richmond, Virginia, USA

    Andrei I. Ivanov

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Xu, G., Deglincerti, A., Paige, J.S., Jaffrey, S.R. (2014). Profiling Lysine Ubiquitination by Selective Enrichment of Ubiquitin Remnant-Containing Peptides. In: Ivanov, A. (eds) Exocytosis and Endocytosis. Methods in Molecular Biology, vol 1174. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0944-5_4

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  • DOI: https://doi.org/10.1007/978-1-4939-0944-5_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0943-8

  • Online ISBN: 978-1-4939-0944-5

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