Label-free measurement of the yeast short chain TAG lipase activity by ESI-MS after one-step esterification[S]
| Autor: | Seung Koo Shin, Hye Jin Ham, Hye-Joo Yoon, Jongcheol Seo |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Electrospray ionization QD415-436 Saccharomyces cerevisiae Biochemistry Catalysis Enzyme catalysis Diglycerides 03 medical and health sciences chemistry.chemical_compound Endocrinology Acyl chloride Methods Lipase Derivatization Triglycerides Diacylglycerol kinase Chromatography biology Esterification urogenital system Hydrolysis Substrate (chemistry) Cell Biology specific activity Lipids Monoacylglycerol lipase 030104 developmental biology chemistry electrospray ionization-mass spectrometry biology.protein lipolysis Monoglycerides chemical derivatization lipids (amino acids peptides and proteins) triacylglycerol |
| Zdroj: | Journal of Lipid Research, Vol 58, Iss 3, Pp 625-631 (2017) |
| Popis: | 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) -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. |
| Databáze: | OpenAIRE |
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