Project description:We present a liquid chromatography/mass spectrometry (LC/MS) method for long-chain and very-long-chain fatty acid analysis and its application to (13)C-tracer studies of fatty acid metabolism. Fatty acids containing 14 to 36 carbon atoms are separated by C(8) reversed-phase chromatography using a water-methanol gradient with tributylamine as ion pairing agent, ionized by electrospray and analyzed by a stand-alone orbitrap mass spectrometer. The median limit of detection is 5 ng/mL with a linear dynamic range of 100-fold. Ratios of unlabeled to (13)C-labeled species are quantitated precisely and accurately (average relative standard deviation 3.2% and deviation from expectation 2.3%). In samples consisting of fatty acids saponified from cultured mammalian cells, 45 species are quantified, with average intraday relative standard deviations for independent biological replicates of 11%. The method enables quantitation of molecular ion peaks for all labeled forms of each fatty acid. Different degrees of (13)C-labeling from glucose and glutamine correspond to fatty acid uptake from media, de novo synthesis, and elongation. To exemplify the utility of the method, we examined isogenic cell lines with and without activated Ras oncogene expression. Ras increases the abundance and alters the labeling patterns of saturated and monounsaturated very-long-chain fatty acids, with the observed pattern consistent with Ras leading to enhanced activity of ELOVL4 or an enzyme with similar catalytic activity. This LC/MS method and associated isotope tracer techniques should be broadly applicable to investigating fatty acid metabolism.
Project description:More than seventy-five isotypes of α-1-antitrypsin (AAT) have been described. To assess risks associated with AAT deficiency, isotype identification is necessary. Isoelectric focusing (IEF) is traditionally used for isotype differentiation, however, IEF has limited scope since it is a manual procedure that is not suitable for automation, and antitrypsin variants must differ in net charge in order to be resolved. In comparison, mass spectrometric assays are easily automated and offer a more complete solution for characterization of proteins. To capitalize on these advantages, we have developed a qualitative top-down liquid chromatography-mass spectrometry (LC-MS) method for selective phenotyping of AAT. This technique requires no sample pretreatment, and has the potential for use in routine clinical diagnostics. We have validated our LC-MS results against both DNA sequencing and IEF. Thus far, this method has identified the AAT variants PLowell, S and Z, as well as unique fragments shared by different M alleles. Its high selectivity is indirectly illustrated by the detection of a variant carrying the amino acid substitution p.Ala308Ser, which cannot be visualized by IEF.
Project description:LC-MS/MS method to determine hydrophobic N-alkyloxy substituted amidines: N-(2-ethylhexyloxy)pyridine-2-carboximidamide, N-(2-ethylhexyloxy)pyridine-3-carboximidamide, N-(2-ethylhexyloxy)pyridine-4-carboximidamide, N-decyloxy pyridine-2-carboximidamide, N-decyloxypyridine-3-carboximidamide and N-decyloxypyridine-4-carboximidamide was developed and validated in terms of linearity, precision and accuracy. The developed method was successfully applied to monitor and control the synthesis process. The experimental data points indicated that the straight chain alkyl bromide reacted most rapidly than branched alkyl bromide and the enhancement of the reaction efficiency strongly depended on reaction temperature.