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.
Highlights
Monophosphorylation of S19 is promoted with low CaMMLCK content and short reaction duration (
The lyophilised smooth muscle tissues are typically stored at À80 C so that multiple myography samples can be analyzed with batch processing
The multiple reaction monitoring (MRM)-mass spectrometry (MS) method was previously assessed in a head-to-head comparison with the Phos-tag SDS-PAGE method for determination of LC20 phosphorylation stoichiometry [7]
Summary
Reactions are initiated by the sequential addition of MgCl2 – ATP solution and MLCK, vortexed gently to ensure complete mixing of constituents, incubated at 30 C, and terminated by addition of EDTA/. The extraction of proteins from smooth muscle tissue is most effective following lyophilisation (>16 h). Smooth muscle tissue can be quenched by immersion in 10% (w/v) TCA, 10 mM DTT in acetone (ice-cold). This procedure inactivates protein kinase and protein phosphatase activities and preserves LC20 phosphorylation status. Take care to ensure the small tissue strip remains immersed in extraction buffer until it is completely solubilized.
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