title: Detection of choline and phosphatidic acid (PA) catalyzed by phospholipase D (PLD) using MALDI-QIT-TOF/MS with 9-aminoacridine matrix.
abstract: Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), the most abundant phospholipids of plasma membrane, resulting in the production of choline and phosphatidic acid (PA). Choline is a precursor of the neurotransmitter acetylcholine, whereas PA functions as an intracellular lipid mediator of diverse biological functions. For assessing PLD activity in vitro, PLD-derived choline has been often analyzed with radioactive or non-radioactive methods. In this study, we have developed a new method for detecting choline and PA with MALDI-QIT-TOF/MS by using 9-aminoacridine as a matrix. The standard calibration curves showed that choline and PA could be detected with linearity over the range from 0.05 and 1 pmol, respectively. Importantly, this method enables the concomitant detection of choline and PA as a reaction product of PC hydrolysis by PLD2 proteins. Thus, our simple and direct method would be useful to characterize the enzymatic properties of PLD, thereby providing insight into mechanisms of PLD activation.
authors.affiliation: a Graduate School of Life and Environmental Sciences , University of Tsukuba , Tsukuba , Japan.
authors.forename: Kyung-Eui
authors.initials: KE
authors.lastname: Park
authors.forename: Jun-Dal
authors.initials: JD
authors.lastname: Kim
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Nagashima
authors.forename: Koichiro
authors.initials: K
authors.lastname: Kako
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Daitoku
authors.forename: Motoki
authors.initials: M
authors.lastname: Matsui
authors.forename: Gwi Gun
authors.initials: GG
authors.lastname: Park
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Fukamizu
mesh_terms: D000585:Aminacrine / Q000378:metabolism*; D055162:Biocatalysis; D002794:Choline / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D006868:Hydrolysis; D057230:Limit of Detection; D010712:Phosphatidic Acids / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D010712:Phosphatidic Acids; D000585:Aminacrine; D010739:Phospholipase D; D002794:Choline
keywords: MALDI-QIT-TOF/MS analysis; choline; phosphatidic acid; phosphatidylcholine hydrolysis; phospholipase D activity