Abstract
Due to the key role of posttranslational modifications (PTMs) such as lysine acetylation in numerous signaling and regulatory pathways, the ability to identify novel PTMs and to quantify their abundances provides invaluable information for understanding these signaling networks. Currently, mass spectrometry (MS) arguably serves as the most high-throughput and unbiased platform for studying PTMs. Here we detail experimental and analytical procedures for the characterization of lysine acetylation on proteins in general and on histones in particular, which are among the most highly modified proteins in eukaryotic cells.
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References
Tolkovsky AM, Wyttenbach A (2009) Differential phosphoprotein labeling (DIPPL) using 32P and 33P. Methods Mol Biol 527:21–29
Archuleta AJ, Stutzke CA, Nixon KM, Browning MD (2011) Optimized protocol to make phosphor-specific antibodies that work. Methods Mol Biol 717:69–88
Steinberg TH, Agnew BJ, Gee KR, Leung WY, Goodman T, Schulenberg B, Hendrickson J, Beechem JM, Haugland RP, Patton WF (2003) Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology. Proteomics 3:1128–1144
Villén J, Gygi SP (2008) The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nat Protoc 3:1630–1638
Norris KL, Lee J, Yao T (2009) Acetylation goes global: the emergence of acetylation biology. Sci Signal 2:76
Choudhary C, Kumar C, Gnad F, Nielsen ML, Rehman M, Walther TC, Olsen JV, Mann M (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325:834–840
Huttlin EL, Jedrychowski MP, Elias JE, Goswami T, Rad R, Beausoleil SA, Villén J, Haas W, Sowa ME, Gygi SP (2010) A tissue-specific atlas of mouse protein phosphorylation and expression. Cell 143:1174–1189
Mann M, Jensen ON (2003) Proteomic analysis of post-translational modifications. Nat Biotechnol 21:255–261
Zee BM, Levin RS, DiMaggio PA, Garcia BA (2010) Global turnover of histone post-translational modifications and variants in human cells. Epigenet Chromatin 3:22
Olsen JV, de Godoy LM, Li G, Macek B, Mortensen P, Pesch R, Makarov A, Lange O, Horning S, Mann M (2005) Parts per million mass accuracy on an orbitrap mass spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics 4:2010–2021
Acknowledgment
BMZ is supported by the NSF Graduate Research Fellowship Program and BAG gratefully acknowledges support from a National Science Foundation (NSF) Early Faculty CAREER award, an NIH Innovator award (DP2OD007447) from the Office of the Director, NIH, and an American Society for Mass Spectrometry research award sponsored by the Waters Corporation. We also thank all members of the Garcia lab for helpful discussion during the composition of this chapter.
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Zee, B.M., Garcia, B.A. (2013). Validation of Protein Acetylation by Mass Spectrometry. In: Hake, S., Janzen, C. (eds) Protein Acetylation. Methods in Molecular Biology, vol 981. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-305-3_1
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DOI: https://doi.org/10.1007/978-1-62703-305-3_1
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