Myoblast Phosphoproteomics as a Tool to Investigate Global Signaling Events During Myogenesis
Protein phosphorylation is a universal covalent chemical modification of amino acids involved in a large number of biological processes including cell signaling, metabolism, proliferation, differentiation, survival/death, ageing, and many more. Regulation of protein phosphorylation is essential in myogenesis and indeed, when the enzymatic activity of protein kinases is distrupted in myoblasts, myogenesis is affected. In this chapter we describe a method to profile the phosphoproteome of myoblasts using mass spectrometry. Phosphate groups are labile and easily lost during the processing of samples for mass spectrometry. Thus, effective methods to enrich for phosphopeptides from protein extracts have been developed. Here, we discuss and present in detail two such methods that we routinely employ. These methods are based on a sample enrichment step performed on titanium dioxide matrices followed by label-free tandem mass spectrometry and semi-quantitation.
Key wordsMyogenesis Phosphoproteomics Myoblasts Muscle stem cells Label-free proteomics Phosphorylation
This work was supported by a PhD studentship to FKJ, GEH and SF from the Biological and Biotechnology Research Council, UK and by a Wellcome Trust ISSF and a Marie Curie IEF to AP.
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