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.

Gas-phase intermolecular phosphate transfer within a phosphohistidine phosphopeptide dimer.

The hydrogen bonds and electrostatic interactions that form between the protonated side chain of a basic residue and the negatively charged phosphate of a phosphopeptide can play crucial roles in governing their dissociation pathways under low-energy collision-induced dissociation (CID). Understanding how phosphoramidate (i.e. phosphohistidine, phospholysine and phosphoarginine), rather than phosphomonoester-containing peptides behave during CID is paramount in investigation of these problematic species by tandem mass spectrometry. To this end, a synthetic peptide containing either phosphohistidine (pHis) or phospholysine (pLys) was analyzed by ESI-MS using a Paul-type ion trap (AmaZon, Bruker) and by traveling wave ion mobility-mass spectrometry (Synapt G2-Si, Waters). Analysis of the products of low-energy CID demonstrated formation of a doubly 'phosphorylated' product ion arising from intermolecular gas-phase phosphate transfer within a phosphopeptide dimer. The results are explained by the formation of a homodimeric phosphohistidine (pHis) peptide non-covalent complex (NCX), likely stabilized by the electrostatic interaction between the pHis phosphate group and the protonated C-terminal lysine residue of the peptide. To the best of our knowledge this is the first report of intermolecular gas-phase phosphate transfer from one phosphopeptide to another, leading to a doubly phosphorylated peptide product ion.