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Assessment of HDACi-Induced Acetylation of Nonhistone Proteins by Mass Spectrometry. | LitMetric
Posttranslational acetylation of lysine residues has been discovered as multifaceted regulatory modification for various nuclear, cytoplasmic, and mitochondrial proteins. The implementation of high-resolution and high-throughput mass spectrometry (MS) approaches has led to the identification of a hitherto underappreciated, large number of acetylation sites for a broad spectrum of cellular proteins. In this chapter, we describe a comprehensive protocol for the purification of an in vivo-acetylated, ectopically expressed, FLAG-epitope tagged nonhistone protein through immunoprecipitation (IP). The protocol also covers the sample preparation by SDS-PAGE, proteolytic digestion, and the analysis by LC-ESI MS. The success of this methodology, however, strongly depends on the physico-chemical properties of the respective protein(s) and the quality of selected peptide mass spectra.
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