Application of reverse-phase HPLC to quantify oligopeptide acetylation eliminates interference from unspecific acetyl CoA hydrolysis
© Evjenth et al; licensee BioMed Central Ltd. 2009
Published: 4 August 2009
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Volume 3 Supplement 6
© Evjenth et al; licensee BioMed Central Ltd. 2009
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.
Acetylation of proteins is a common protein modification that occurs either in the N-terminal α amino group (Nα-acetylation) or the ε amino group of lysine residues (Nε-acetylation). The corresponding acetylation reactions are catalysed by Nα-acetyltransferases (NATs) or histone acetyltransferases/lysine acetyltransferases (HATs/KATs), respectively [1, 2].
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 . The generation of radioactively labelled oligopeptides is monitored by a filter-binding assay and liquid scintillation counting . This assay is very sensitive , but due to the use of radioactivity, the assay represents potential environmental and health risks and it is therefore relatively demanding to perform due to safety precautions. The other commonly used method is a spectrophotometric assay that continuously measures the amount of CoASH generated by the acetyltransferase reactions . The CoASH is determined by a coupled enzyme system using either α-ketoglutarate dehydrogenase or pyruvate dehydrogenase. The CoASH dependent oxidation of α-ketoglutarate or pyruvate is coupled to the reduction of NAD+ to NADH, which is determined spectrophotometrically at 340 nm. This assay is relatively inexpensive and can be performed with standard spectrophotometric equipment. A disadvantage of both methods is the difficulty to detect whether an oligopeptide substrate contains more than one lysine target residue.
Using the production of CoA as the basis for measuring acetyltransferase activity is linked to another potentially severe cause of error. In nearly all KAT assays, measuring the Nε-acetylation of lysines, a substantial amount of acetyl CoA will spontaneously react with the ε amino group on lysine side chains, making it necessary to design proper controls to correct for this effect .
In addition, using the CoA-NADH coupled enzyme assay, it is not possible to use CoA as inhibitor to study acetyltransferase catalytic mechanism and, similarly to the filter assay, when more than one lysine target is present in the oligopeptide substrate, detailed acetylation site specificity can not be studied. To determine Km and Vmax values, the initial reaction rate must be determined under conditions giving linear initial reaction rates over the range of substrate concentration used, normally the substrate conversion should not exceed 10 – 15% . Furthermore, build-up of high concentrations of products may cause product inhibition. It is therefore of interest to obtain detailed information both on substrate consumption and product production. The radioactivity-based filter assay and the coupled-enzyme assay do not provide information on product consumption and detailed control experiments must be added. In the present article, we present a simple method for studying oligopeptide acetylation, using reverse phase HPLC detecting acetylated oligopeptides, in addition to CoASH. This method uses semi-automated HPLC-technology providing a fast, sensitive and highly reproducible assay for protein acetyltransferases. The instrument records the UV spectrum between 200 and 300 nm, thereby enabling to monitor acetyl CoA and CoA (260 nm) and the peptide substrate and acetylated oligopeptides (215 nm) simultaneously. In addition, a radioactive flow detector may be connected to detect 14C/3H-acetyl-oligopeptides to increase sensitivity, if required.
After the peptide separation using reverse phase HPLC, the relevant absorbance peaks are integrated and the corresponding areas are converted to amounts of product formed. The continuous recording of the UV spectra allows for each peak to be evaluated for the maximum absorbance wavelength which can be used to determine the purity of each absorbance signal.
Since the oligopeptide separation is performed in buffers containing acetonitrile and TFA, the HPLC method is also fully compatible with subsequent analytical procedures such as mass spectrometry to determine the site of modification.
Oligopeptides used as substrates in this study
[H]MLGTE GG RWGRPVGRRRRPVRVYP [OH]*
hnRNP H; (P31943)
[H]MLAL ISR RWGRPVGRRRRPVRVYP [OH]*
[H]MLGTG PA RWGRPVGRRRRPVRVYP [OH]*
[H]MLGP EGG RWGRPVGRRRRPVRVYP [OH]*
hnRNP F; (P52597)
The catalytic subunit of the human NatC complex; hNaa30p (earlier known as hMak3), a NAT acetyltransferase, was used to establish the reverse phase HPLC method. hNAA30 was cloned into the prokaryotic expression system pETM-MBP (originally obtained from G. Stier – EMBL, Heidelberg, Germany) and expressed in E. coli. The cloning, expression and purification were performed as described .
Prior to sample injection, the column was equilibrated for 5 minutes (0.35 ml/min flow rate) with buffer A (5% acetonitrile (ACN) and 0.1% trifluoracetic acid (TFA)). After sample injection, the column was washed for 8 minutes with 2% elutionbuffer B (90% ACN, 0.1% TFA). The oligopeptides were then eluted employing a 40 minutes linear gradient from 2% to 40% buffer B. The column was then rinsed with 95% buffer B for 5 minutes. Finally, a 2 minutes linear gradient to 2% buffer B was performed.
80 nM of purified MBP-hNaa30p with 200 μM 1MLGTE-RRR24 oligopeptide and 300 μM acetyl CoA in acetylation buffer (50 mM Tris-HCl (pH 8.5), 10% Glycerol, 1 mM EDTA) were incubated for 60 minutes at 37°C. Samples were collected after 0, 10, 20, 30, and 60 minutes incubation and analyzed by reverse phase HPLC.
To determine the Km oligopeptides, 80 nM of purified MBP-hNaa30p was incubated with varying concentrations of oligopeptides (30 to 350 μM) and 300 μM acetyl CoA in acetylation buffer for 30 minutes at 37°C. When determining the Km acetyl CoA, 300 μM of 1MLAL-RRR24 peptide was used in combination with varying ‘concentrations of acetyl CoA (4 to 40 µM). The enzyme reactions were stopped by adding TFA to final concentrations of 1% (v/v). The amounts of acetylated oligopeptides were determined based on the absorbance at 215 nm, while the production of CoA was determined by using the absorbance at 260 nm. The steady-state enzyme kinetic parameters were calculated by nonlinear regression analysis using the SigmaPlot Technical Graphing Software (SPSS Inc.) The normality tests for all Km determinations were passed with value > 0.8.
To verify the elution time for the acetylated oligopeptides, we conducted a time dependent acetylation assay by incubating purified MBP-hNaa30p (80 nM) with the oligopeptide 1MLGTE-RRR24 (200 μM) and [1-14C] acetyl CoA (final concentration 300 μM with specific activity 11.2 mCi/mmol). Samples were collected after 0, 10, 20, 30, and 60 minutes, placed on ice and adjusted to 1% TFA.
Comparison of Km oligopeptide and Vmax based on the detection of CoA(260 nm) and the detection of acetylated oligopeptides(215 nm).
Vmax (pmol product * min-1 * pmol hNaa30p-1)
Vmax (pmol product * min-1 * pmol hNaa30p-1)
The radioactivity-based filter assay  and the CoA-NADH coupled enzyme assay  are the most commonly applied methods to study acetyltransferase kinetics and mechanisms. Both these methods suffer from significant drawbacks such as biohazard and non-enzymatic deacetylation of acetyl CoA. To eliminate these problems and to allow us to analyse the acetylated oligopeptide products by mass spectrometry, we developed a method for studying peptide acetylation based on reverse phase HPLC. After acetylation, non-acetylated and acetylated oligopeptides are separated and quantified by integrating the respective elution peaks.
Since the first residues of the substrates seem to be most important for enzyme specificity , we designed peptides that deviated only within the 7 first N-terminal positions. The next 17 amino acids that are indicated by 'RRR' are identical in all peptides and resemble the sequence of Adrenocorticotropic hormone (ACTH) (Amino acid no. 8 to 24), but all Lys residues were replaced with Arg to minimize aberrant N-ε acetylation. The separation of peptides were carried out with 0.1% TFA in the HPLC buffers, making the peptide residues highly protonated. The positively charged Arg residues facilitate peptide solubility and separation by reverse phase HPLC. The N-terminal acetylation substitutes a positive charge by a hydrophobic group, causing the acetylated oligopeptides to be separated from the non acetylated form due to stronger interaction with the Nucleosil C18 HD matrix, resulting in increased elution time. Several acetylation assays with other Nα-acetyltransferases acetylating oligopeptides containing more hydrophobic residues, showed that the acetylated form of these oligopeptides also could be efficiently separated with the reverse phase Nucleosile C18HD column. This indicates that commercially available oligopeptide substrates, composed of the endogenous amino acids, can be used as substrates with this detection method.
The unacetylated and acetylated oligopeptide 1MLGTE-RRR24 was separated by more than 3 minutes. Even when using high amounts of oligopeptides, >30 nmols, leading to a widening of the peaks, an efficient separation of unacetylated and acetylated oligopeptides was achieved. If necessary, the separation of oligopeptides can be further enhanced by optimising the elution profile.
The sensitivity of the detection method was determined by injecting different amount of oligopeptides and acetyl CoA and quantifying the resulting absorption profiles. This showed a clear linear trend for both substrates, spanning from 0.5 to 5 nmol of acetyl CoA and from 1 to 10 nmol of oligopeptide. The coefficient of determination (R2) was above 0.97 for both substrates. Our experience with this acetylation assay is that even at very high amounts of oligopeptides, up to 30 nmol, the increase in absorbance at 215 nm is linear with coefficient of determination above 0.97 (data not shown).
The reproducibility of the method was analysed by calculating the standard deviation (S.D.) from three independent experiments. This was done for the sensitivity determination and the calculation of the kinetic constants. The result demonstrated that the reverse phase HPLC method is highly reproducible when analysing acetylation based on the detection of acetylated oligopeptides with the Abs 215 nm signal.
In conclusion, we have established a robust and highly reproducible method for studying oligopeptide acetylation. With new semi-automated reverse phase HPLC technology, we show that both substrates, acetyl CoA and unacetylated oligopeptides, and enzyme products, CoA and acetylated oligopeptides can be detected and quantified in the same experiment. This allows for increased control over substrate conversion and product generation forming a solid basis for data intepretation. Importantly, the assay is easy to perform and automation reduces sample handling.
We thank L. Vikebø, M. Algroy and N. Glomnes for technical assistance.
This article has been published as part of BMC Proceedings Volume 3 Supplement 6, 2009: Proceedings of the 2007 and 2008 Symposia on Protein N-terminal Acetylation. The full contents of the supplement are available online at http://www.biomedcentral.com/1753-6561/3?issue=S6
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.