Tools and protocol for quantification of myosin phosphorylation with MRM-MS

Justin A MacDonald; Annegret Ulke-Lemée; Mona Chappellaz; Hayden Segboer

doi:10.1016/j.mex.2018.05.004

Tools and protocol for quantification of myosin phosphorylation with MRM-MS

MethodsX. 2018 May 17:5:466-474. doi: 10.1016/j.mex.2018.05.004. eCollection 2018.

Authors

Justin A MacDonald¹, Annegret Ulke-Lemée¹, Mona Chappellaz¹, Hayden Segboer¹

Affiliation

¹ Department of Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB, T2N 4Z6, Canada.

Abstract

The phosphorylation of myosin regulatory light chain (LC20) at Thr18 and Ser19 is positively correlated with tension development in smooth muscle tissue, and the molar stoichiometry of LC20 phosphorylation is commonly profiled as a measure of smooth muscle contractility. We provide details for a newly applied multiple reaction monitoring (MRM)-mass spectrometry (MS) method for the quantification of LC20 phosphorylation at Thr18 and Ser19. This MRM-MS method provides a robust alternative to antibody-based detection systems (such as Phos-Tag SDS-PAGE) for the quantification of LC20 phosphorylation.

Keywords: Artery; Intestine; LC20; MLC; MRM-MS myosin phosphorylation assay; Myosin regulatory light chain; Phosphorylation; Quantitation; Serine 19; Smooth muscle; Stoichiometry; Targeted proteomics; Threonine 18.