Tools and protocol for quantification of myosin phosphorylation with MRM-MS
Justin A MacDonald1, Annegret Ulke-Lemée1, Mona Chappellaz1
1Department of Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB, T2N 4Z6, Canada.
Methodsx
|January 10, 2019
Summary
Quantifying myosin regulatory light chain (LC20) phosphorylation at Thr18 and Ser19 using mass spectrometry offers a reliable method. This technique accurately measures smooth muscle contractility, serving as an alternative to antibody-based assays.
Area of Science:
- Biochemistry
- Physiology
- Analytical Chemistry
Background:
- Myosin regulatory light chain (LC20) phosphorylation at Thr18 and Ser19 correlates with smooth muscle tension.
- Quantifying LC20 phosphorylation is crucial for assessing smooth muscle contractility.
Purpose of the Study:
- To detail a novel multiple reaction monitoring (MRM)-mass spectrometry (MS) method for quantifying LC20 phosphorylation at Thr18 and Ser19.
- To establish MRM-MS as a robust alternative to existing antibody-based detection methods.
Main Methods:
- Multiple reaction monitoring (MRM) coupled with mass spectrometry (MS).
- Quantification of phosphorylated LC20 at specific sites (Thr18 and Ser19).
Main Results:
- The developed MRM-MS method accurately quantifies LC20 phosphorylation at Thr18 and Ser19.
- This method provides a robust and reliable alternative to antibody-based techniques like Phos-Tag SDS-PAGE.
Conclusions:
- MRM-MS is a viable and robust technique for quantifying LC20 phosphorylation.
- This method enhances the assessment of smooth muscle contractility through precise phosphorylation profiling.
Keywords:
ArteryIntestineLC20MLCMRM-MS myosin phosphorylation assayMyosin regulatory light chainPhosphorylationQuantitationSerine 19Smooth muscleStoichiometryTargeted proteomicsThreonine 18More Related Videos
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