title: 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.
authors.affiliation: The Biomedical Research Centre, ∥The Centre for Blood Research, University of British Columbia , Vancouver, BC V6T 1Z3, Canada.
authors.forename: Cheng-Cheng
authors.initials: CC
authors.lastname: Zhang
authors.forename: Ru
authors.initials: R
authors.lastname: Li
authors.forename: Honghui
authors.initials: H
authors.lastname: Jiang
authors.forename: Shujun
authors.initials: S
authors.lastname: Lin
authors.forename: Jason C
authors.initials: JC
authors.lastname: Rogalski
authors.forename: Kate
authors.initials: K
authors.lastname: Liu
authors.forename: Juergen
authors.initials: J
authors.lastname: Kast
mesh_terms: D001792:Blood Platelets; D002478:Cells, Cultured; D056148:Chromatography, Reverse-Phase; D020549:GTP Phosphohydrolase Activators / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D015539:Platelet Activation / Q000502:physiology; D020033:Protein Isoforms / Q000032:analysis* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
chemical_list: D020549:GTP Phosphohydrolase Activators; D010446:Peptide Fragments; D020033:Protein Isoforms
keywords: Small GTPases; activity; isoforms; multiplexing; platelet activation; quantification; targeted proteomics