New Isotopic Labelling Methodology and its Application in Phosphoproteomics

The kinetics of protein phosphorylation and dephosphorylation are tightly controlled by specific kinases and phosphatases; disturbances are often disease-causing. Phosphorylation kinetics are normally monitored using radioactive isotopes of phosphorus, or by using stop-flow techniques. Approaches using mass spectrometry are severely limited by the lack of a stable isotope of phosphorus (other than 31P). The principal aim of this study is to develop a new method to incorporate 18O label into phosphorylation sites of phosphoproteins with a view of applying this method to enhance the detection of phosphorylation by mass spectrometry and to analyze the phosphorylation kinetics of proteins.Aurora-A kinase was selected to explore the possibility of using 18O-labelling to monitor phosphorylation kinetics. The kinase is well characterized, phosphorylated both in human cells and when expressed in recombinant form in E. coli and it contributes to development of some cancers when deregulated.Applying different mass spectrometric approaches resulted in the identification of 19 phosphorylation sites of Aurora-A including five new sites. Using H3P18O4 as a label donor to incorporate 18O into Aurora-A phosphorylation sites showed partial and inconsistent label incorporation. Alternatively, H218O was used to investigate the possibility of label incorporation. Preliminary results, however, showed high complex data which hampered precise identification of phosphopeptides and their labelling state.The labelling experiment was then redesigned in which induction took place in label free medium to allow the light version of the kinase to accumulate, before chasing with 18O label. This design successfully introduced fully labelled P18O3 into Aurora-A phosphorylation sites. LC ESI Q-ToF analysis of 18O labelled Aurora-A sample isolated according to this protocol identified 30 phosphopeptides showing label incorporation, which is double the number of phosphopeptides identified by MASCOT using the same MS analysis. The method was also used to investigate phosphorylation kinetics of Aurora-A. The results suggested differential regulation of phosphorylation sites of Aurora-A as some sites showed early phosphorylation while others were phosphorylated at later stages. Overall, a new approach was developed for enhanced detection of phosphorylation sites and analysis of phosphorylation kinetics.