Kinase-substrate analysis via diaPASEF® phosphoproteomics
Webinar Overview
Duration: 16 minutes
The S. cerevisiae (yeast) ISR1 gene encodes a putative kinase with no specific function ascribed, however over expression of ISR1 is lethal. To determine the function and substrates of this kinase, we conducted diaPASEF® phosphoproteomics experiment in both ISR1 knockout and ISR1 expressing cells. From this analysis we quantified 8,879 phosphopeptides. Of these sites, we identify 4 sites of phosphorylation on Gfa1, one of which (S332) was up-regulated by nearly 200-fold in the presence if the ISR1 kinase. Alanine mutations of this residue, as well as adjacent S/T residues, rescued the lethality associated with ISR1 over expression. These results suggest that Gfa1 is a bonafide substrate of ISR1, and implicates a role for ISR1 in the hexosamine biosynthesis pathway. In short, we demonstrate the utility of diaPASEF® phosphoproteomics to rapidly and confidently determine the substrates and function for uncharacterized kinases.
Speaker
Danielle Swaney
For Research Use Only. Not for use in clinical diagnostic procedures.