Limits...
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.


Related in: MedlinePlus

Reverse phase HPLC absorbance profile at 215 nm for the separation of acetylated and non-acetylated peptides. A; The oligopeptide 1MLGTE-RRR24 (200 μM) was incubated with acetyl CoA (300 μM) and purified MBP-hNaa30p (80 nM) in acetylation buffer for 60 minutes at 37°C. Samples were collected at indicated time points and analysed with reverse phase HPLC. The resulting absorbance profile at 215 nm indicate good separation of unacetylated ('a') and acetylated oligopeptides ('b'). B; An expanded version of the absorbance profile for the formation of acetylated oligopeptide. A clear time dependent increase in the absorption signal is observed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2722098&req=5

Figure 2: Reverse phase HPLC absorbance profile at 215 nm for the separation of acetylated and non-acetylated peptides. A; The oligopeptide 1MLGTE-RRR24 (200 μM) was incubated with acetyl CoA (300 μM) and purified MBP-hNaa30p (80 nM) in acetylation buffer for 60 minutes at 37°C. Samples were collected at indicated time points and analysed with reverse phase HPLC. The resulting absorbance profile at 215 nm indicate good separation of unacetylated ('a') and acetylated oligopeptides ('b'). B; An expanded version of the absorbance profile for the formation of acetylated oligopeptide. A clear time dependent increase in the absorption signal is observed.

Mentions: 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. Then we tested whether unacetylated and acetylated oligopeptides could be efficiently separated. We incubated an oligopeptide that is expected be a good hNaa30p substrate [7]; 1MLGTE-RRR24 (200 μM) and 300 μM acetyl CoA with 80 nM of purified MBP-hNaa30p in the acetylation buffer. The sample was incubated at 37°C with aliquots collected after 0, 10, 20, 30, and 60 minutes (see Methods). From the elution profile at Abs 215 nm, we observed, as expected, a large amount of unmodified oligopeptide (Figure 2A, peak annotated 'a') and an additional peak with delayed elution time which increased with enzyme reaction time (Figure 2A and 2B, peak annotated 'b'). The changes in absorbance profiles recorded at 260 nm, detecting CoA and acetyl CoA in the same run, showed the same tendencies as the Abs 215 nm signals (Figure 3A and 3B). We observed a large excess of acetyl CoA (Figure 3A, peak annotated 'b') and an additional peak that increases during the course of the reaction (Figure 3A and 3B, peak annotated 'a'). The elution times of acetyl CoA and CoA were determined by injecting 3 nmol of CoA or acetyl CoA and recording the resulting absorbance profiles. This analysis showed that CoA and acetyl CoA eluted after 6 minutes and 9 minutes and 30 seconds, respectively, peaks 'a' and 'b' in Figure 3A.


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)

Reverse phase HPLC absorbance profile at 215 nm for the separation of acetylated and non-acetylated peptides. A; The oligopeptide 1MLGTE-RRR24 (200 μM) was incubated with acetyl CoA (300 μM) and purified MBP-hNaa30p (80 nM) in acetylation buffer for 60 minutes at 37°C. Samples were collected at indicated time points and analysed with reverse phase HPLC. The resulting absorbance profile at 215 nm indicate good separation of unacetylated ('a') and acetylated oligopeptides ('b'). B; An expanded version of the absorbance profile for the formation of acetylated oligopeptide. A clear time dependent increase in the absorption signal is observed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Reverse phase HPLC absorbance profile at 215 nm for the separation of acetylated and non-acetylated peptides. A; The oligopeptide 1MLGTE-RRR24 (200 μM) was incubated with acetyl CoA (300 μM) and purified MBP-hNaa30p (80 nM) in acetylation buffer for 60 minutes at 37°C. Samples were collected at indicated time points and analysed with reverse phase HPLC. The resulting absorbance profile at 215 nm indicate good separation of unacetylated ('a') and acetylated oligopeptides ('b'). B; An expanded version of the absorbance profile for the formation of acetylated oligopeptide. A clear time dependent increase in the absorption signal is observed.
Mentions: 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. Then we tested whether unacetylated and acetylated oligopeptides could be efficiently separated. We incubated an oligopeptide that is expected be a good hNaa30p substrate [7]; 1MLGTE-RRR24 (200 μM) and 300 μM acetyl CoA with 80 nM of purified MBP-hNaa30p in the acetylation buffer. The sample was incubated at 37°C with aliquots collected after 0, 10, 20, 30, and 60 minutes (see Methods). From the elution profile at Abs 215 nm, we observed, as expected, a large amount of unmodified oligopeptide (Figure 2A, peak annotated 'a') and an additional peak with delayed elution time which increased with enzyme reaction time (Figure 2A and 2B, peak annotated 'b'). The changes in absorbance profiles recorded at 260 nm, detecting CoA and acetyl CoA in the same run, showed the same tendencies as the Abs 215 nm signals (Figure 3A and 3B). We observed a large excess of acetyl CoA (Figure 3A, peak annotated 'b') and an additional peak that increases during the course of the reaction (Figure 3A and 3B, peak annotated 'a'). The elution times of acetyl CoA and CoA were determined by injecting 3 nmol of CoA or acetyl CoA and recording the resulting absorbance profiles. This analysis showed that CoA and acetyl CoA eluted after 6 minutes and 9 minutes and 30 seconds, respectively, peaks 'a' and 'b' in Figure 3A.

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.


Related in: MedlinePlus