Abstract
The dynamic nature of protein posttranslational modification (PTM) allows cells to rapidly respond to changes in their environment, such as nutrition, stress, or signaling. Lysine residues are targets for several types of modifications, including methylation, ubiquitination, and various acylation groups, especially acetylation. Currently, one of the best methods for identification and quantification of protein acetylation is immunoaffinity enrichment in combination with high-resolution mass spectrometry. As we are using a relatively novel and comprehensive mass spectrometric approach, data-independent acquisition (DIA), this protocol provides high-throughput, accurate, and reproducible label-free PTM quantification. Here we describe detailed protocols to process relatively small amounts of mouse liver tissue that integrate isolation of proteins, proteolytic digestion into peptides, immunoaffinity enrichment of acetylated peptides, identification of acetylation sites, and comprehensive quantification of relative abundance changes for thousands of identified lysine acetylation sites.
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Acknowledgments
This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (R24 DK085610 E.V. and R01 DK090242, Goetzman/B.S.). We acknowledge support from the NIH shared instrumentation grant for the TripleTOF system at the Buck Institute (1S10 OD016281, Gibson). J.G.M. was supported by a National Institutes of Health grant (T32 AG000266). We thank Dr. Davalyn Powell for editing the manuscript.
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Schilling, B., Meyer, J.G., Wei, L., Ott, M., Verdin, E. (2019). High-Resolution Mass Spectrometry to Identify and Quantify Acetylation Protein Targets. In: Brosh, Jr., R. (eds) Protein Acetylation. Methods in Molecular Biology, vol 1983. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9434-2_1
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DOI: https://doi.org/10.1007/978-1-4939-9434-2_1
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