PROJECT SUMMARY/ABSTRACT Requested here are funds to purchase FPLC instrumentation that will support NIH-funded research in an R01 grant (1R01GM145035-01A1) entitled, “Elucidating the Molecular Basis of Cellular Metal Stress by using Mass Spectrometry-Based Proteomic Methods.” The instrument to be purchased is a Cytiva AKTA Pure Fast Protein Liquid Chromatography System (FPLC). The requested funds will be used to purchase the instrument and associated attachments necessary for its function (approximately $85,000). Access to this instrument is critical to the success of research being performed. The on-going and proposed studies related to the above grant build on preliminary and recently published results from this collaborative team showing the utility of a pulse proteolysis mass spectrometry method developed in MPI Fitzgerald’s laboratory to identify protein targets of Cu in E. coli, establishing these proteomic methodologies for the study of metal-protein interactions. These preliminary and recently published results have identified protein targets that are now being isolated and purified for rigorous mechanistic studies. The fplc instrumentation is critical for the rigor of protein purification needed for functional and spectroscopic studies. The overall objective of the parent grant is to identify proteins that are functionally affected when cells experience stress induced by exposure to excess levels of Zn, Cu, and Ag, as a non-native surrogate of Cu(I). This objective is being met by using a powerful combination of mass spectrometry-based proteomic methods to address four specific aims: 1) Determine global profiles of protein stability changes as a function of cellular metal overload and metal deficiency across bacterial, fungal, and human cancer cells; 2) Establish a mechanistic basis linking differential stability of Aim 1 proteins to function; 3) Identify the relative sensitivity of proteins across the proteome to misfolding induced by Cu, Ag, and Zn binding; and 4) Establish a biophysical basis for understanding the relative sensitivity of proteins to metal-induced misfolding. Two of the four above specific aims (i.e., Aims 2, and 4) require access to the requested instrument.