Chapter title |
Assessment of HDACi-Induced Acetylation of Nonhistone Proteins by Mass Spectrometry
|
---|---|
Chapter number | 23 |
Book title |
HDAC/HAT Function Assessment and Inhibitor Development
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-6527-4_23 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6525-0, 978-1-4939-6527-4
|
Authors |
Martin Wieczorek, Karl-Heinz Gührs, Thorsten Heinzel |
Abstract |
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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Unspecified | 1 | 33% |
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