Peptidyl-hydroxyglycine alpha-amidating lyase (PAL) is one of the two enzymes which are necessary for biosynthetic post-translational processing of glycine-extended peptide prohormones into active carboxy-terminal amidated peptide hormones. Several amidated peptides are produced by tumor cells and act on those cells to stimulate growth. We are developing tools to measure the enzymes which produce amidated peptides, to identify cells undergoing this autocrine growth stimulation. Reported assays for amidating enzymes generally involve radiolabelled substrates and laborious extraction or chromatographic methods for analysis of enzymatic reaction product [1]. This poster describes a simple LC-MS assay for PAL which we have developed using a modified tripeptide substrate.
Syntheses: PAL has been shown to act on relatively lipophilic substrates containing an S-alpha-hydroxy carboxylic acid moiety. Commencing with the dipeptide L-Phe-L-Phe-carboxyamide we synthesized the N-trinitrophenyl analogue (TNP-FF#, the expected product of the PAL reaction) and from that TNP-FF-alphahydroxyglycine (TNP-FF-aHOG, the PAL substrate). Both peptides were purified by HPLC. We have not yet been able to resolve the synthesized racemic mixture of TNP-FF-HOGs.
LC-MS: A 5µL Rheodyne loop was overfilled (2x), and sample was injected onto an ABI Aquapore 300Å 7m 1x100 mm column. Mobile phase was 20 mM ammonium acetate, pH 5, with 60% AcCN. Flow rate used was 40µL/min. Samples were manually injected every 3.5 minutes. A PE-SCIEX API III plus was operated using RAD v2.4 . The method pauses for one minute then collects data for 2.5 minutes with a dwell time of 500 msec/ion. Data was analyzed using the MacQuan v1.2 software. Data for two ions was collected in Q1MI (i.e. SIM) mode. TNP-FF# was recorded as the ammonium adduct (calculated m/z 540.2), and TNP-FF-HOG as the m+1 isotope of the ammonium adduct (calculated m/z 615.2) to prevent detector overload. Immediately before each cycle of operation a mix of the two analytes was analyzed in Q1 scan mode (500-650 amu) with a 0.1 amu step to ascertain the highest intensity ion mass (always between 539.9 and 540.3 for TNP-FF#) which was then used in the method. Standards of appropriate varying concentrations of TNP-FF# were included with each batch of samples. Overfilling the injector loop provides very good reproducibility without the need for an internal standard. The standards can be fitted to a quadratic curve over almost three orders of magnitude (0.1 pmol to 50 pmol). Sensitivity for TNP-FF# was approx. 1 pmol in a 20 µL injection. Sensitivity was not reduced in the presence of large amounts of TNP-FF-HOG, up to 1000 pmol (a situation analogous to that of a standard substrate-excess enzymatic reaction condition).
Assay method: The PAL enzymatic assay is carried out for 1 hour at 37°C with duplicate 5µL enzyme samples in a final volume of 20µL 50mM ammonium acetate pH 4.5 with 200uM TNP-FF-aHOG. The reaction was stopped by addition of 50µL acetonitrile and cooling the sample on ice. Activities parallel those determined by a method using a substrate mix prepared from radio-iodinated N-acetylTyrPheGly and a peptidylglycine hydroxylase. The ease of synthesis of this PAL substrate, and the simple and quick LC-MS analysis, will greatly assist our studies on the biochemistry of peptide amidation in tumors and cancer cell lines [1].