Probing lysine acetylation in proteins: strategies, limitations, and pitfalls of in vitro acetyltransferase assays. Mol Cell Proteomics 4

The acetylation of proteins at specific lysine residues by acetyltransferase enzymes has emerged as a posttransla-tional modification of high biological impact. Although ly-sine acetylation in histone proteins is an integral part of the histone code the acetylation of a multitude of non-histone proteins was recently recognized as a regulatory signal in many cellular processes. New substrates of acetyltrans-ferase enzymes are continuously identified, and the analy-sis of acetylation sites in proteins is increasingly performed by mass spectrometry. However, the characterization of lysine acetylation in proteins using mass spectrometric techniques has some limitations and pitfalls. The non-en-zymatic cysteine acetylation especially can result in false-positive identification of acetylated proteins. Here we dem-onstrate the application of various mass spectrometric techniques such as matrix-assisted laser desorption/ioni-zation and electrospray ionization mass spectrometry for the analysis of protein acetylation. We describe diverse combinations of biochemical methods useful to map the acetylation sites in proteins and discuss their advantages and limitations. As an example, we present a detailed analysis of the acetylation of the HIV-1 transactivator of transcription (Tat) protein, which is known to be acety-lated in vivo by the acetyltransferases p300 and p300/ CBP-associated factor (PCAF). Molecular & Cellula