Assessment of HDACi-Induced Acetylation of Nonhistone Proteins by Mass Spectrometry

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.

Acetylation-induced proteasomal degradation of the activated glucocorticoid receptor limits hormonal signaling

“…Detection of acetylation was performed as in. 56 , 57 Briefly, for global acetylation of GR, cells expressing FLAG- or Ty1- tagged GR were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA [pH 8.0], 1 mM EGTA [pH 8.0], 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors (100 mM PMSF, aprotinin, leupeptin), phosphatase inhibitors (10 mM sodium orthovanadate, 0.5 mM sodium fluoride) and lysine deacetylase inhibitors including 1 μM trichostatin A (TSA) and 10 mM nicotinamide (NAM). Cell suspensions were sonicated with a Branson sonifier and cleared by high-speed centrifugation.…”

“…Peptides were subjected to nano-liquid electrospray ionized tandem mass spectrometry (LC-ESI-MS/MS) using an LQ Orbitrap XL ETD (Thermo Fischer Scientific) as described in. 57 The mass spectrometer was set up to scan the full spectrum ranging from 300 to 2000 Da followed by three MS/MS spectra after CID (collision induced dissociation) fragmentation of multiple charged precursors. Peptide sequences were attributed to the spectra using ProteomeDiscoverer software (v1.4, Thermo Fischer Scientific) in combination with Mascot (v2.4 Matrix Science) and the Swiss-Prot database ( www.uniprot.org ).…”

The stability and antiapoptotic activity of Bm30K‐3 can be improved by lysine acetylation in the silkworm, Bombyx mori

“…Lysine acetylation was first observed in histones and is a modification that is catalyzed by histone acetyltransferases (HATs) and histone deacetyltransferases (HDACs) and regulates the function of chromatin (Allfrey, Faulkner, & Mirsky, ). With the development of proteomics technologies, increasing numbers of nonhistone proteins have been shown to also be acetylated and deacetylated by HATs and HDACs, respectively (Glozak, Sengupta, Zhang, & Seto, ; Wieczorek, Guhrs, & Heinzel, ). Acetylation can regulate the functions of nonhistone proteins by altering protein stability, subcellular localization, and protein–nucleic acid/protein–protein interactions (Narita, Weinert, & Choudhary, ).…”