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

Evjenth R, Hole K, Ziegler M, Lillehaug JR - BMC Proc (2009)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, University of Bergen, N-5020 Bergen, Norway. rune.evjenth@mbi.uib.no

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.

No MeSH data available.


Standard curves of increasing amount of the substrate 1MLGP-RRR24 and acetyl CoA analysed by reverse phase HPLC. A; Five different amounts of the oligopeptide 1MLGP-RRR24, diluted in acetylation buffer, were analysed by reverse phase HPLC. The resulting absorption signals at 215 nm were quantified. Each amount were analysed three times and error bars indicate S.D. B; Five different amounts of acetyl CoA, diluted in acetylation buffer, were analysed by reverse phase HPLC. The resulting absorption signals at 260 nm were quantified. Each amount were analysed three times and error bars indicate S.D.
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Figure 4: Standard curves of increasing amount of the substrate 1MLGP-RRR24 and acetyl CoA analysed by reverse phase HPLC. A; Five different amounts of the oligopeptide 1MLGP-RRR24, diluted in acetylation buffer, were analysed by reverse phase HPLC. The resulting absorption signals at 215 nm were quantified. Each amount were analysed three times and error bars indicate S.D. B; Five different amounts of acetyl CoA, diluted in acetylation buffer, were analysed by reverse phase HPLC. The resulting absorption signals at 260 nm were quantified. Each amount were analysed three times and error bars indicate S.D.

Mentions: The sensitivity of the HPLC based analysis was studied by injecting different amounts of oligopeptide and recording the resulting Abs 215 nm signal. The sensitivity for acetyl CoA was determined by injecting different amounts of acetyl CoA and recording the resulting Abs 260 nm signal. A linear correlation between the absorbance signals and the amount of substrates added was observed (Figure 4A and 4B). We noted that 0.5 nmol was the lower limit for reliable quantification of oligopeptides at 215 nm, which corresponded to 5 μM in a reaction volume of 100 μl.


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

Evjenth R, Hole K, Ziegler M, Lillehaug JR - BMC Proc (2009)

Standard curves of increasing amount of the substrate 1MLGP-RRR24 and acetyl CoA analysed by reverse phase HPLC. A; Five different amounts of the oligopeptide 1MLGP-RRR24, diluted in acetylation buffer, were analysed by reverse phase HPLC. The resulting absorption signals at 215 nm were quantified. Each amount were analysed three times and error bars indicate S.D. B; Five different amounts of acetyl CoA, diluted in acetylation buffer, were analysed by reverse phase HPLC. The resulting absorption signals at 260 nm were quantified. Each amount were analysed three times and error bars indicate S.D.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2722098&req=5

Figure 4: Standard curves of increasing amount of the substrate 1MLGP-RRR24 and acetyl CoA analysed by reverse phase HPLC. A; Five different amounts of the oligopeptide 1MLGP-RRR24, diluted in acetylation buffer, were analysed by reverse phase HPLC. The resulting absorption signals at 215 nm were quantified. Each amount were analysed three times and error bars indicate S.D. B; Five different amounts of acetyl CoA, diluted in acetylation buffer, were analysed by reverse phase HPLC. The resulting absorption signals at 260 nm were quantified. Each amount were analysed three times and error bars indicate S.D.
Mentions: The sensitivity of the HPLC based analysis was studied by injecting different amounts of oligopeptide and recording the resulting Abs 215 nm signal. The sensitivity for acetyl CoA was determined by injecting different amounts of acetyl CoA and recording the resulting Abs 260 nm signal. A linear correlation between the absorbance signals and the amount of substrates added was observed (Figure 4A and 4B). We noted that 0.5 nmol was the lower limit for reliable quantification of oligopeptides at 215 nm, which corresponded to 5 μM in a reaction volume of 100 μl.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, University of Bergen, N-5020 Bergen, Norway. rune.evjenth@mbi.uib.no

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.

No MeSH data available.