4.6 Article

Label-free measurement of the yeast short chain TAG lipase activity by ESI-MS after one-step esterification

Journal

JOURNAL OF LIPID RESEARCH
Volume 58, Issue 3, Pages 625-631

Publisher

ELSEVIER
DOI: 10.1194/jlr.D074385

Keywords

chemical derivatization; triacylglycerol; lipolysis; specific activity; electrospray ionization-mass spectrometry

Funding

  1. National Research Foundation of Korea [NRF-2013R1A1A2009426]
  2. Brain Korea 21 Plus program [21A20131312508]

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Triacylglycerol (TAG) lipases hydrolyze ester bonds in TAG and release diacylglycerol (DAG), monoacylglycerol (MAG), and FA. We present a one-step chemical de-rivatization method for label-free quantification of a mixture of TAG, DAG, and MAG following lipase assay by ESI-MS. Because the ionization efficiencies of TAG, DAG, and MAG are not identical, lipase reaction products, DAG and MAG, are derivatized to TAG species by esterifying their hydroxyl groups using acyl chloride, whose acyl chain contains one less (or one more)-CH2 group than that of substrate TAG. This resulted in three TAG species that were separated by 14 Da from one another and exhibited similar ion responses representing their molar amounts in the mass spectra. A good linear correlation was observed between peak intensity ratios and molar ratios in calibration curve. This method enables simultaneous quantification of TAG, DAG, and MAG in lipase assay and, in turn, allows stoichiometric determination of the concentrations of FAs released from TAG and DAG separately. By applying this strategy to measure both TAG and DAG lipolytic activities of the yeast Tgl2 lipase, we demonstrated its usefulness in studying enzymatic catalysis, as lipase enzymes often show dissimilar activities toward these lipids.

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