Development and Application of a Quantitative Multiplexed Small GTPase Activity Assay Using Targeted Proteomics
| Autor: | Ru Li, Kate Liu, Shujun Lin, Honghui Jiang, Jason C. Rogalski, Chengcheng Zhang, Juergen Kast |
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| Rok vydání: | 2015 |
| Předmět: |
Blood Platelets
Proteomics Gene isoform Cell signaling GTPase Biology Biochemistry Multiplexing Western blot Tandem Mass Spectrometry medicine Humans Protein Isoforms Small GTPase Platelet activation Cells Cultured Chromatography Reverse-Phase medicine.diagnostic_test Selected reaction monitoring Reproducibility of Results General Chemistry Platelet Activation Peptide Fragments Cell biology Isotope Labeling GTP Phosphohydrolase Activators |
| Zdroj: | Journal of Proteome Research. 14:967-976 |
| ISSN: | 1535-3907 1535-3893 |
| DOI: | 10.1021/pr501010v |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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