Abstract
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.
Highlights
Acetylation of proteins is a common protein modification that occurs either in the N-terminal α amino group (Nαacetylation) or the ε amino group of lysine residues (Nεacetylation)
We present a simple method for studying oligopeptide acetylation, using reverse phase HPLC detecting acetylated oligopeptides, in addition to CoASH
The HPLC system To establish the chromatographic procedure, first, the elution times for unmodified oligopeptides were determined by injecting 3 nmol of pure oligopeptides diluted in the acetylation buffer on the HPLC system
Summary
Acetylation of proteins is a common protein modification that occurs either in the N-terminal α amino group (Nαacetylation) or the ε amino group of lysine residues (Nεacetylation). The important biological functions of protein acetylation have promoted extensive functional studies of different acetyltransferases to determine their kinetic properties, substrate specificities and catalytic mechanisms. For most of the enzymatic analyses, two different kinds of acetyl transfer assays are used. One uses radioactively labelled acetyl CoA as substrate [3]. The generation of radioactively labelled oligopeptides is monitored by a filter-binding assay and liquid scintillation counting [3]. This assay is very sensitive [4], but due to the use of radioactivity, the assay represents potential environmental and health risks and it is relatively demanding to perform due to safety precautions. The other commonly used method is a (page number not for citation purposes)
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