Edited by John M. Denu This work was supported by grants from the National Science Foundation (MCB-1714569), the UCLA Academic Senate Faculty Research Program, the Life Extension Foundation, Inc., and the Elizabeth and Thomas Plott Chair in Gerontology of the UCLA Longevity Center (to S. G. C.). The authors declare that they have no conflicts of interest with the contents of this article. The dataset containing amino acid sequences of all identified crystallin protein fragments from this publication is available in the MassIVE database under accession code MSV000084028. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Tables S1-S9 and Figs. S1 and S2.

Small GTPases are a family of key signaling molecules that are ubiquitously expressed in various types of cells. Their activity is often analyzed by western blot, which is limited by its multiplexing capability, the quality of isoform-specific antibodies, and the accuracy of quantification. To overcome these issues, a quantitative multiplexed small GTPase activity assay has been developed. Using four different binding domains, this assay allows the binding of up to 12 active small GTPase isoforms simultaneously in a single experiment. To accurately quantify the closely related small GTPase isoforms, a targeted proteomic approach, i.e., selected/multiple reaction monitoring, was developed, and its functionality and reproducibility were validated. This assay was successfully applied to human platelets and revealed time-resolved coactivation of multiple small GTPase isoforms in response to agonists and differential activation of these isoforms in response to inhibitor treatment. This widely applicable approach can be used for signaling pathway studies and inhibitor screening in many cellular systems.