Development and Application of a Quantitative Multiplexed Small GTPase Activity Assay Using Targeted Proteomics

Abstract

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.

Platelets in Healthy and Disease States: From Biomarkers Discovery to Drug Targets Identification by Proteomics

“…Moreover, a targeted quantitative proteomic approach using LC-MRM MS has been applied to study small GTPase isoforms at different time points from human platelets in response to several agonists [ 138 ]. In particular, the active small GTPases were precipitated using a specific resin, separated by Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE), and tryptic digested with the addition of heavy isotope labelled peptides for the LC-MRM MS analysis.…”

“…Moreover, a targeted quantitative proteomic approach using LC-MRM MS has been successfully used to monitor in parallel small GTPase isoforms at different time points from human platelets in response to several agonists [ 138 ].…”

A Targeted Quantitative Proteomic Approach Assesses the Reprogramming of Small GTPases during Melanoma Metastasis

“…In this context, Halvey et al (13) developed an LC-MRM method for quantifying wild-type and mutant K-RAS proteins in cultured cancer cells and pancreatic cyst fluids after enrichment of low-molecular weight (20–25 kDa) proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, Zhang et al (14) described the use of MRM in combination with SDS-PAGE-based enrichment of proteins in the molecular weight range of 15–25 kDa to measure simultaneously the activities of 12 small GTPases after affinity enrichment using the GTPase-binding domains of four effector proteins. Building upon these previous studies, we developed an SDS-PAGE fractionation coupled with LC-MRM workflow for targeted quantification of small GTPases at the entire proteome scale.…”

Targeted Proteomics Comes to the Benchside and the Bedside: Is it Ready for Us?

“…For example, it is difficult to perform GTPase activity measurement for different but closely related forms due to the lack of specific antibodies. An MRM assay to measure 12 different isoforms of GTPase simultaneously in the presence of agonists or inhibitors was developed and utilized . Thus, there are a significant number of studies available now, which have been reviewed in other articles, that provide us with a complete picture of current applications of targeted proteomics in different areas of biology .…”