title: Label-free measurement of the yeast short chain TAG lipase activity by ESI-MS after one-step esterification.
abstract: Triacylglycerol (TAG) lipases hydrolyze ester bonds in TAG and release diacylglycerol (DAG), monoacylglycerol (MAG), and FA. We present a one-step chemical derivatization 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) -CH2group 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.
authors.affiliation: Bio-Nanotechnology Center, Department of Chemistry, Pohang University of Science and Technology, Pohang, Kyungbuk 37673, Republic of Korea.
authors.forename: Hye Jin
authors.initials: HJ
authors.lastname: Ham
authors.affiliation: Bio-Nanotechnology Center, Department of Chemistry, Pohang University of Science and Technology, Pohang, Kyungbuk 37673, Republic of Korea.
authors.forename: Jongcheol
authors.initials: J
authors.lastname: Seo
authors.affiliation: Bio-Nanotechnology Center, Department of Chemistry, Pohang University of Science and Technology, Pohang, Kyungbuk 37673, Republic of Korea hjyoon@postech.ac.kr skshin@postech.ac.kr.
authors.forename: Hye-Joo
authors.initials: HJ
authors.lastname: Yoon
authors.affiliation: Bio-Nanotechnology Center, Department of Chemistry, Pohang University of Science and Technology, Pohang, Kyungbuk 37673, Republic of Korea hjyoon@postech.ac.kr skshin@postech.ac.kr.
authors.forename: Seung Koo
authors.initials: SK
authors.lastname: Shin
mesh_terms: D002384:Catalysis; D004075:Diglycerides / Q000737:chemistry / Q000378:metabolism; D004951:Esterification; D006868:Hydrolysis; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification*; D008055:Lipids / Q000737:chemistry*; D050178:Monoglycerides / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014280:Triglycerides / Q000737:chemistry / Q000378:metabolism
chemical_list: D004075:Diglycerides; D008055:Lipids; D050178:Monoglycerides; D014280:Triglycerides; D008049:Lipase
keywords: chemical derivatization; electrospray ionization-mass spectrometry; lipolysis; specific activity; triacylglycerol