Mass Spectrometry for Proteomic Studies of Zinc Homeostasis

The investigators will develop a novel technology using mass spectrometry coupled with a residue-specific stable isotope labeling technique that has broad applications in various aspects of proteomic research. They have been able to incorporate stable isotope labeled amino acid precursors into proteins in a residue-specific manner. This labeling technique introduces a characteristic mass splitting pattern that can be readily recognized and be used to determine the existence as well as the number of labeled residues. The researchers have demonstrated that this technique could be employed to improve the identification of unknown proteins and the detection of protein modifications. In this R21 project, they will further develop this technique for high-confidence, high-throughput identification of protein post-translational modifications. Moreover, this technique will be applied to the accurate quantification of protein expression and modification levels, computational tools will be developed to automate data analysis for this specialized MS. The different tools will then be integrated as a complete bioinformatics package for automated data analysis for this stable isotope labeling mass spectrometry. Proteomic studies of zinc ion homeostasis in S. cerevisiae will be carried out in concert with the methodology development. Zinc is critical for human health and has been implicated in different diseases. A protein network has been known to be involved in zinc homeostasis, but certain critical components are missing. New technologies are needed to achieve a more complete understanding. Data obtained on the known zinc-responsive proteins will first be used to validate the proteomic platform. More importantly, the investigators will employ this proteomic approach to examine the zinc responsive alternations in the yeast proteome. They will focus on the plasma membrane proteins in this R21 project. Zrtlp and Zrt2p are two known zinc transporters on the plasma membrane. A comparison of membrane protein expression levels will be made between wild type and zrtl zrt2 double deletion strains to identify additional unidentified zinc transporter(s). They will also characterize the post-translational modifications of membrane proteins, which will help to elucidate the mechanism by which cells sense the zinc concentration in their environment.