An optimised MALDI-TOF assay for phosphatidylcholine-specific phospholipase C

Nabangshu Sharma; Ries J. Langley; Chatchakorn Eurtivong; Euphemia Leung; Ryan Joseph Dixon; Emily K. Paulin; Shaun W. P. Rees; Lisa I. Pilkington; David Barker; Jóhannes Reynisson; Ivanhoe K. H. Leung

doi:10.1039/D0AY02208J

An optimised MALDI-TOF assay for phosphatidylcholine-specific phospholipase C†

Nabangshu Sharma,

^a Ries J. Langley,

^bc Chatchakorn Eurtivong,

^de Euphemia Leung,

^bcf Ryan Joseph Dixon,^a Emily K. Paulin,^a Shaun W. P. Rees,^a Lisa I. Pilkington,

^a David Barker,

^agh Jóhannes Reynisson

^ai and Ivanhoe K. H. Leung

‡*^abg

Author affiliations

* Corresponding authors

^a School of Chemical Sciences, The University of Auckland, Private Bag 92019, Victoria Street West, Auckland 1142, New Zealand
E-mail: i.leung@auckland.ac.nz

^b Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, Victoria Street West, Auckland 1142, New Zealand

^c Department of Molecular Medicine and Pathology, School of Medical Sciences, The University of Auckland, Private Bag 92019, Victoria Street West, Auckland 1142, New Zealand

^d Program in Chemical Sciences, Chulabhorn Graduate Institute, Chulabhorn Royal Academy, Bangkok 10210, Thailand

^e Center of Excellence on Environmental Health and Toxicology (EHT), Commission on Higher Education (CHE), Ministry of Education, Bangkok 10400, Thailand

^f Auckland Cancer Society Research Centre, School of Medical Sciences, The University of Auckland, Private Bag 92019, Victoria Street West, Auckland 1142, New Zealand

^g Centre for Green Chemical Science, The University of Auckland, Private Bag 92019, Victoria Street West, Auckland 1142, New Zealand

^h The MacDiarmid Institute for Advanced Materials and Nanotechnology, Victoria University of Wellington, PO Box 600, Wellington 6140, New Zealand

ⁱ School of Pharmacy and Bioengineering, Keele University, Staffordshire ST5 5BG, UK

Abstract

The Bacillus cereus phosphatidylcholine-specific phospholipase C (PC-PLC_Bc) is an enzyme that catalyses the hydrolysis of phosphatidylcholines into phosphocholine and 1,2-diacylglycerols. PC-PLC_Bc has found applications in both the food industry and in medicinal chemistry. Herein, we report our work in the development and optimisation of a matrix assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry-based assay to monitor PC-PLC_Bc activity. The use of one-phase and two-phase reaction systems to assess the inhibition of PC-PLC_Bc with different structural classes of inhibitors was compared. We also highlighted the advantage of our assay over the commonly used commercially available Amplex Red assay. This method will also be applicable to work on the activity and inhibition of other phospholipases.

Analytical Methods

An optimised MALDI-TOF assay for phosphatidylcholine-specific phospholipase C†

Abstract

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An optimised MALDI-TOF assay for phosphatidylcholine-specific phospholipase C

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