# Quantifying small GTPase activation status using a novel fluorescence HPLC-based assay

## TL;DR

A new sensitive method called Fluor-HPLC is developed to measure the activation states of small GTPases, which are important in cellular signaling and diseases like cancer.

## Contribution

A novel fluorescence HPLC-based assay is introduced for precise quantification of small GTPase activation states.

## Key findings

- Fluor-HPLC successfully quantified guanine nucleotide-binding states of small GTPases at endogenous levels.
- The method elucidated RHEB and HRAS activation in response to extracellular stimuli.
- Fluor-HPLC provided insights into KRAS activation dynamics in tumor tissues and therapeutic effectiveness.

## Abstract

Small GTPases play crucial roles in cellular signaling pathways, with their activation states tightly regulated between GDP-bound inactive and GTP-bound active forms. Dysregulation of these nucleotide-binding states, such as in oncogenic RAS, is implicated in diseases like cancer. Accurately quantifying these states in cells is thus crucial for deciphering their functional roles and regulatory mechanisms. However, current methods do not fully meet the necessary sensitivity and versatility, limiting their effectiveness in small GTPase analysis. Here, we present a highly sensitive HPLC-based assay with fluorescence detection (Fluor-HPLC), enabling precise quantification of guanine nucleotide-binding states in small GTPases. Applying this technique, we successfully quantified the guanine nucleotide-binding states of small GTPases at their endogenous expression levels. We demonstrated the utility of Fluor-HPLC by elucidating RHEB and HRAS activation in response to extracellular stimuli. Furthermore, integration of Fluor-HPLC with syngeneic mouse models provided insights into KRAS activation dynamics in tumor tissues and evaluated the effectiveness of targeted therapeutics. Overall, this versatile method paves the way for investigating activation states and regulatory mechanisms of various small GTPases, potentially accelerating our understanding of their roles in cellular processes and disease pathogenesis.