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This article is part of the supplement: Proceedings of the 2007 and 2008 Symposia on Protein N-terminal Acetylation

Open Access Proceedings

Application of reverse-phase HPLC to quantify oligopeptide acetylation eliminates interference from unspecific acetyl CoA hydrolysis

Rune Evjenth1, Kristine Hole123, Mathias Ziegler1 and Johan R Lillehaug1*

Author Affiliations

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

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BMC Proceedings 2009, 3(Suppl 6):S5  doi:10.1186/1753-6561-3-S6-S5

Published: 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.