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Generation of acetyllysine antibodies and affinity enrichment of acetylated peptides

Abstract

Lysine acetylation has emerged as one of the major post-translational modifications, as indicated by its roles in chromatin remodeling, activation of transcription factors and, most recently, regulation of metabolic enzymes. Identification of acetylation sites in a protein is the first essential step for functional characterization of acetylation in physiological regulation. However, the study of the acetylome is hindered by the lack of suitable physical and biochemical properties of the acetyl group and existence of high-abundance acetylated histones in the cell, and needs a robust method to overcome these problems. Here we present protocols for (i) using chemically acetylated ovalbumin and synthetic acetylated peptide to generate a pan-acetyllysine antibody and a site-specific antibody to Lys288-acetylated argininosuccinate lyase, respectively; (ii) using subcellular fractionation to reduce highly abundant acetylated histones; and (iii) using acetyllysine antibody affinity purification and mass spectrometry to characterize acetylome of human liver tissue. The entire characterization procedure takes 2–3 d to complete.

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Figure 1: Schematic diagram of enrichment of acetylated peptides from human liver.
Figure 2: Acetylated proteins have higher molecular weight.
Figure 3: Monitoring of enriched peptides by RP-LC.
Figure 4: Identification of multiacetylated forms of acetylated histone peptides.
Figure 5: MS spectra of enriched acetylated peptides.

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References

  1. Driessen, H.P., de Jong, W.W., Tesser, G.I. & Bloemendal, H. The mechanism of N-terminal acetylation of proteins. CRC Crit. Rev. Biochem. 18, 281–325 (1985).

    Article  CAS  PubMed  Google Scholar 

  2. Yang, X.J. & Seto, E. HATs and HDACs: from structure, function and regulation to novel strategies for therapy and prevention. Oncogene 26, 5310–5318 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Phillips, D.M. The presence of acetyl groups of histones. Biochem. J. 87, 258–263 (1963).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Allfrey, V.G., Faulkner, R. & Mirsky, A.E. Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Proc. Natl. Acad. Sci. USA 51, 786–794 (1964).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Grunstein, M. Histone acetylation in chromatin structure and transcription. Nature 389, 349–352 (1997).

    Article  CAS  PubMed  Google Scholar 

  6. Paolinelli, R., Mendoza-Maldonado, R., Cereseto, A. & Giacca, M. Acetylation by GCN5 regulates CDC6 phosphorylation in the S phase of the cell cycle. Nat. Struct. Mol. Biol. 16, 412–420 (2009).

    Article  CAS  PubMed  Google Scholar 

  7. Gu, W. & Roeder, R.G. Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 90, 595–606 (1997).

    Article  CAS  PubMed  Google Scholar 

  8. Marzio, G. et al. E2F family members are differentially regulated by reversible acetylation. J. Biol. Chem. 275, 10887–10892 (2000).

    Article  CAS  PubMed  Google Scholar 

  9. Yang, Y. et al. Acetylation of FoxO1 activates Bim expression to induce apoptosis in response to histone deacetylase inhibitor depsipeptide treatment. Neoplasia 11, 313–324 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kim, S.C. et al. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol. Cell 23, 607–618 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Zhao, S. et al. Regulation of cellular metabolism by protein lysine acetylation. Science 327, 1000–1004 (2006).

    Article  Google Scholar 

  12. Choudhary, C. et al. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325, 834–840 (2009).

    Article  CAS  PubMed  Google Scholar 

  13. Qiang, L., Xiao, H., Campos, E.I., Ho, V.C. & Li, G. Development of a PAN-specific, affinity-purified anti-acetylated lysine antibody for detection, identification, isolation, and intracellular localization of acetylated protein. J. Immunoassay Immunochem. 26, 13–23 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Hirschey, M.D., Shimazu, T., Huang, J.Y. & Verdin, E. Acetylation of mitochondrial proteins. Methods Enzymol. 457, 137–147 (2009).

    Article  CAS  PubMed  Google Scholar 

  15. Takemura, R. et al. Increased microtubule stability and alpha tubulin acetylation in cells transfected with microtubule-associated proteins MAP1B, MAP2 or tau. J. Cell Sci. 103 (Pt 4): 953–964 (1992).

    Article  CAS  PubMed  Google Scholar 

  16. Gaertig, J. et al. Acetylation of lysine 40 in alpha-tubulin is not essential in Tetrahymena thermophila. J. Cell Biol. 129, 1301–1310 (1995).

    Article  CAS  PubMed  Google Scholar 

  17. Lu, B., Iwuoha, E.I., Smyth, M.R. & O'Kennedy, R. Effects of acetonitrile on horseradish peroxidase (HRP)-anti HRP antibody interaction. Biosens. Bioelectron. 12, 619–625 (1997).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We are grateful to members of the Fudan MCB lab, especially those who contributed to the development of these techniques. We thank Y. Zhen of Shanghai Genomics Inc for generating antibodies and providing detailed information. This work is supported by the 985 Program of the Chinese Ministry of Education, by the State Key Development Program of Basic Research of China (2009CB918401, 2009CB918600 and 2006CB806700), by the National High Technology Research and Development Program of China (2006AA02A308), and by grants from the Chinese National Science Foundation (30971485/C0706, 30600112 and 30871255), the Shanghai Key Project for Basic Research, China (09JC1402300, 07PJ14011 and 08JC1400900).

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K.-L.G., Y. X. and S.Z. designed the protocols; Y.L., W.Y. and S.Z. carried out the experiments; K.-L.G. and S. Z. wrote the paper.

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Correspondence to Kun-Liang Guan or Shimin Zhao.

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The authors declare no competing financial interests.

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Guan, KL., Yu, W., Lin, Y. et al. Generation of acetyllysine antibodies and affinity enrichment of acetylated peptides. Nat Protoc 5, 1583–1595 (2010). https://doi.org/10.1038/nprot.2010.117

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