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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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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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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DOI: https://doi.org/10.1038/nprot.2010.117
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