Development and Application of a New Platform for Measuring Phospholipase A2 Activity in Cells Using Mass Spectrometry

Abstract

Members of the phospholipase A2 (PLA2) superfamily hydrolyze fatty acids from the sn‐2 position of membrane phospholipids. PLA2‐mediated release of n‐3 and n‐6 polyunsaturated fatty acids (PUFAs) is a highly regulated and critical step in producing eicosanoids and docosanoids. These signaling molecules play key roles in a variety of important processes including inflammation, onset of premature labor, and neural degenerative diseases. Thus, a better understanding of how PLA2 functions in cells may uncover new therapeutic avenues and lead to potent pharmacological agents. Though a rapid in vitro assay of PLA2 function has been recently published, one barrier to progress in this field is the lack of methods for determining PLA2 activity in cells. To address this issue, we have developed a new platform for measuring PLA2 activity ex vivo. Enriching cells with natural or deuterated PUFAs followed by mass spectrometry allows for tracking of hydrolysis at the sn‐2 position by both the disappearance of substrate phospholipids and the appearance of product PUFAs. An optimized cell culture procedure and lipid analysis workflow allow for significantly improved throughput. We anticipate that this platform will not only lead to a better understanding of PLA2 regulation and function in cells, but also allow for rapid screening of inhibitor candidates.Support or Funding InformationSupported by NIH grant GM20501