Ubiquitin (Ub), an ancient signaling protein, is chemically attached to other proteins in the cell to regulate their function. The Ub system is critical for normal cellular function, and malfunctions of the Ub system have been linked to many different human maladies, including cancer, Alzheimer's and Parkinson's diseases, and mental retardation. Mass spectrometry (MS) has emerged as an extremely powerful tool to study complex biological samples. Using a combination of MS and a novel software suite (SUMmOn) that we created, my colleagues and I developed the first automated approach for the identification and characterization of Ub and ubiquitin-like proteins. Here, using these tools and other advanced molecular biology techniques, we propose to conduct the first complete characterization of the Ub system in the model organism S. cerevisiae (budding yeast). High density protein-protein interaction mapping will be used to characterize interacting partners for all of the Ub system enzymes, and proteins containing Ub binding motifs. These data will be used to inform similar studies in the more complex mammalian system. In this way, we will gain a much better understanding of how the complex and critically important Ub system is regulated, and learn more about the processes that are in turn regulated by this critically important protein.