Abstract
AbstractTo further understand ubiquitin‐binding proteins, we have developed a panel of monoubiquitin affinity resins linked through six different positions: residues 6, 11, 29, 48, 63, and 76. Each resin bound a different subset of yeast proteins. MALDI‐TOF MS analysis of the eluted proteins identified several of these proteins. Adding excess free ubiquitin competes for the binding of specific ubiquitin‐binding proteins. Thus, putative monoubiquitin‐binding proteins could be identified by this method. Analysis of yeast protein with this panel of resins demonstrates clear differences in the protein‐binding pattern, depending on what ubiquitin residue is coupled to the resin. The results suggested that certain proteins show a preference for binding to different faces of ubiquitin. Sap185, an effector of the Sit4 protein phosphatase, has been identified as a putative ubiquitin‐binding protein that binds to a face of ubiquitin other than that containing the hydrophobic patch. The combined affinity chromatography and mass spectrometry approach is a powerful tool for identifying ubiquitin‐binding proteins.
Published Version
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