Due to the importance of reversible phosphorylation in virtually all aspects of cell function and development, there exists a need to develop better methods to identify and quantify changes in the phosphorylation states of proteins on a proteome-wide level. In this R21 project, we will develop and apply new approaches, termed phosphopeptide isotope coded affinity tags (PhIAT), for obtaining proteome-wide identification and precise measurements of differences in the phosphorylation states of the proteins extracted from p53+/+ mouse cortical neurons. Our approach will utilize proteome-wide stable isotope and biotin labeling of phosphopeptides to enable high affinity isolation of phosphopeptides. We will use data-dependent tandem mass spectrometry (MS/MS) and Fourier transform ion cyclotron resonance mass spectrometry (FTICR/MS) to identify phosphorylated peptides that can function as accurate phosphopeptide mass tags (APMTs) to uniquely identify phosphorylated proteins. The approach will provide for high affinity isolation of phosphopeptides, be at least 3 orders of magnitude more sensitive than existing 2-D PAGE methodologies, and be able to rapidly identify and measure relative phosphorylation states for thousands of proteins in a single analysis. We will apply this technology to quantify differences in the relative phosphorylation state of proteins from p53+/+ and p53-/- cortical neurons treated with an apoptotic stimulus. The later phase of this project will develop methods that concomitantly combine PhIAT and ICAT labeling to identify proteins in glutamate or camptothecin treated p53+/+ cortical neurons that undergo changes in either their phosphorylation state or relative abundance compared to non-treated cells. By combining the PhIAT and ICAT strategies on treated p53+/+ neurons, we will be able to identify proteins that undergo a change in phosphorylation without a corresponding change in expression, or vice versa. The development of this capability will ultimately provide the broadest present proteome coverage since changes in protein abundance, as well as changes in protein phosphorylation states will be identifiable in a single experiment.
