This article is part of the supplement: Proceedings of the 2007 and 2008 Symposia on Protein N-terminal Acetylation
Application of reverse-phase HPLC to quantify oligopeptide acetylation eliminates interference from unspecific acetyl CoA hydrolysis
1 Department of Molecular Biology, University of Bergen, N-5020 Bergen, Norway
2 Department of Surgical Sciences, University of Bergen, N-5020 Bergen, Norway
3 Department of Surgery, Haukeland University Hospital, N-5021 Bergen, Norway
BMC Proceedings 2009, 3(Suppl 6):S5 doi:10.1186/1753-6561-3-S6-S5Published: 4 August 2009
Protein acetylation is a common modification that plays a central role in several cellular processes. The most widely used methods to study these modifications are either based on the detection of radioactively acetylated oligopetide products or an enzyme-coupled reaction measuring conversion of the acetyl donor acetyl CoA to the product CoASH. Due to several disadvantages of these methods, we designed a new method to study oligopeptide acetylation. Based on reverse phase HPLC we detect both reaction products in a highly robust and reproducible way. The method reported here is also fully compatible with subsequent product analysis, e.g. by mass spectroscopy. The catalytic subunit, hNaa30p, of the human NatC protein N-acetyltransferase complex was used for N-terminal oligopeptide acetylation. We show that unacetylated and acetylated oligopeptides can be efficiently separated and quantified by the HPLC-based analysis. The method is highly reproducible and enables reliable quantification of both substrates and products. It is therefore well-suited to determine kinetic parameters of acetyltransferases.