Probing Lysine Acetylation in Proteins: Strategies, Limitations, and Pitfalls of in Vitro Acetyltransferase Assays

Wilma Dormeyer,Melanie Ott,Martina Schnölzer

doi:10.1074/mcp.m500047-mcp200

Abstract

The acetylation of proteins at specific lysine residues by acetyltransferase enzymes has emerged as a posttranslational modification of high biological impact. Although lysine 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 acetyltransferase enzymes are continuously identified, and the analysis 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-enzymatic cysteine acetylation especially can result in false-positive identification of acetylated proteins. Here we demonstrate the application of various mass spectrometric techniques such as matrix-assisted laser desorption/ionization 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 acetylated in vivo by the acetyltransferases p300 and p300/CBP-associated factor (PCAF).

Highlights

The acetylation of proteins at specific lysine residues by acetyltransferase enzymes has emerged as a posttranslational modification of high biological impact
The reversible lysine acetylation of histones and nonhistone proteins plays a vital role in the regulation of many cellular processes including chromatin dynamics and transcription [2,3,4,5], gene silencing [6, 7], cell cycle progression (8 –11), apoptosis [12,13,14], differentiation [15,16,17,18,19], DNA replication [20, 21], DNA repair [22,23,24,25,26,27], nuclear import (28 –30), and neuronal repression [31,32,33]
Over 40 transcription factors and 30 other nuclear, cytoplasmic, bacterial, and viral proteins have been shown to be acetylated in vivo [34, 35], and the investigation of protein acetylation continues

Summary

Probing Lysine Acetylation in Proteins

The acetylation of proteins at specific lysine residues by acetyltransferase enzymes has emerged as a posttranslational modification of high biological impact. New substrates of acetyltransferase enzymes are continuously identified, and the analysis of acetylation sites in proteins is increasingly performed by mass spectrometry. We present a detailed analysis of the acetylation of the HIV-1 transactivator of transcription (Tat) protein, which is known to be acetylated in vivo by the acetyltransferases p300 and p300/ CBP-associated factor (PCAF). For exemplification we used the human immunodeficiency virus, type 1 (HIV-1) transactivator of transcription (Tat) protein and peptides from the human histone H4 protein that are known substrates of the acetyltransferases p300 and p300/CBP-associated factor (PCAF) [34]. Diverse combinations of experiments useful to map the acetylation sites in proteins and peptides are presented, and potential limitations and pitfalls in the interpretation of in vivo and in vitro acetylation studies are discussed

EXPERIMENTAL PROCEDURES

GGLGISYGRKKRRQRRRP GGLGISYGRAcKKRRQRRRP

RESULTS AND DISCUSSION