Continuous Electrospray Ionization Mass Spectrometry Assay for Measuring Phospholipase Activity against Liposomes

Phospholipases have diverse roles in lipid and cell membrane biology. In animal venoms, they can have roles as neurotoxins or myotoxins that disrupt the integrity of cell membranes. In this work, we describe a temperature-controlled, continuous electrospray ionization mass spectrometry (ESI-MS) assay for measuring phospholipase A₂ activity against liposomes. The enzyme used in this assay was paradoxin, which is a neurotoxic trimeric phospholipase A₂ from inland taipan snake venom. Previously developed ESI-MS-based phospholipase assays have been discontinuous and analyzed hydrolysis of single lipid molecules by liquid chromatography ESI-MS. In this work, a continuous assay was developed against liposomes, a more complex substrate that more closely reflects the natural substrate for paradoxin. The assay confirmed the requirement for Ca²⁺ and allowed measurement of Michaelis-Menten-type parameters. The use of ESI-MS for lipid detection enabled nuanced insights into the effect of changing assay conditions not only on the enzyme but also on the liposome substrate. Changing the metal ion concentrations did not significantly change the liposomes but did affect enzymatic activity. Increasing temperature did not substantially affect the secondary structure of paradoxin but affected liposome size, resulting in increased enzymatic activity consistent with the disruption of the phosphatidylcholine membrane, increasing accessibility of sn-2 ester bonds. The continuous ESI-MS method described herein can be applied to other enzyme reactions, particularly those which utilize complex lipid substrates.