Related Experiment Video

Updated: Jan 22, 2026

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics
09:18

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Published on: April 17, 2017

10.3K

Intrinsic GTPase Activity of K-RAS Monitored by Native Mass Spectrometry

Zahra Moghadamchargari1, Jamison Huddleston1, Mehdi Shirzadeh1

  • 1Department of Chemistry , Texas A&M University , College Station , Texas 77843 , United States.

Biochemistry
|July 16, 2019
PubMed

Insights

RAS mutations drive cancer by resisting inactivation. This study uses mass spectrometry to reveal that oncogenic K-RAS mutants unexpectedly hydrolyze 2'-deoxy GTP faster than GTP, offering new therapeutic insights.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Oncology

Background:

  • RAS mutations are key drivers in numerous cancers, making them significant therapeutic targets.
  • RAS inactivation occurs via GTP hydrolysis, a process crucial for regulating cell signaling.
  • Cancer-associated RAS mutations often exhibit resistance to GTPase-activating protein (GAP) stimulation, relying solely on intrinsic hydrolysis for inactivation.

Purpose of the Study:

  • To determine the kinetics and transition state thermodynamics of intrinsic GTP hydrolysis for K-RAS and its oncogenic mutants.
  • To investigate the impact of specific mutations (G12C, Q61H, G13D) on K-RAS intrinsic GTPase activity.
  • To explore the heterogeneity of nucleotide binding (2'-deoxy vs. 2'-hydroxy GDP/GTP) in recombinant K-RAS.

Main Methods:

  • High-resolution native mass spectrometry (MS) was employed to directly monitor intrinsic GTPase activity.
  • K-RAS intrinsic GTPase activity was measured by observing the mass loss corresponding to phosphate release from GTP-bound K-RAS.
  • MS-derived kinetic rates were compared with those obtained from established solution-based assays.

Main Results:

  • MS data revealed heterogeneity in nucleotide binding, with both 2'-deoxy and 2'-hydroxy forms of GDP and GTP bound to recombinant K-RAS.
  • The intrinsic GTPase activity of K-RAS was found to have enthalpically and entropically unfavorable transition state thermodynamics.
  • Oncogenic K-RAS mutants (G12C, Q61H, G13D) unexpectedly demonstrated a higher intrinsic hydrolysis rate for 2'-deoxy GTP compared to GTP.

Conclusions:

  • The study provides novel insights into the intrinsic hydrolysis mechanisms of K-RAS and its oncogenic mutants using native MS.
  • The observed thermodynamic properties of K-RAS intrinsic GTPase activity highlight the energetic challenges of inactivation.
  • The unexpected differential hydrolysis rates for 2'-deoxy GTP in mutants suggest potential new avenues for targeted cancer therapies against RAS-driven tumors.

More Related Videos

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics09:09

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Published on: October 13, 2020

5.1K
Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling09:35

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Published on: April 1, 2017

14.5K

Related Concept Videos

Small GTPases - Ras and Rho01:24

Small GTPases - Ras and Rho

Ras and Rho are small monomeric GTPases that act downstream of receptor tyrosine kinase (RTK) and regulate various cellular processes. These GTPases switch between active and inactive states by binding to guanine nucleotides.
Three regulatory proteins control their activity:
5.2K
GTPases and their Regulation02:14

GTPases and their Regulation

Guanine nucleotide-binding proteins (G-proteins), also known as GTPases, are a superfamily of proteins that regulate many cellular processes, such as cell signaling, vesicular transport, and the regulation of cell shape and motility. Mutation or dysfunction of these proteins can lead to disease. There are around 40,000 known G-proteins that can broadly be classified into two groups ‒  small G-proteins consisting of a single domain and large multi-domain G-proteins.
Large G-proteins,...
9.8K
The Ras Gene02:38

The Ras Gene

The Ras-gene-encoded proteins are regulators of signaling pathways controlling cell proliferation, differentiation, or cell survival. The Ras-gene family in humans constitutes three primary members—the HRas, NRas, and KRas. These genes code for four functionally distinct yet closely related proteins—the HRas, NRas, KRas4A, and KRas4B. The involvement of mutant Ras genes in human cancer was first discovered in 1982 and is among the most common causes of human tumorigenesis.
Ras is a...
7.1K
Tandem Mass Spectrometry01:21

Tandem Mass Spectrometry

Tandem mass spectrometry is a technique that uses multiple mass analyzers in series to obtain a higher selectivity and reduce chemical noise during analyte detection. Instruments with multiple analyzers separated by an interaction cell enable secondary fragmentation and selected study of the fragment ions.Secondary fragmentations occur in the interaction cell and can be induced by various factors. Fragmentation induced by collision with inert gases, such as N2, Ar, He, etc., is called...
2.4K
Mass Spectrometry: Overview01:19

Mass Spectrometry: Overview

Mass spectrometry is an analytical technique used to determine the molecular mass and molecular formula of a compound. The basic principle of mass spectrometry is to generate ions from the analyte molecule and measure these ion abundances against their molecular mass. One common type of ionization, known as electron ionization or EI, bombards the analyte molecules in the gas phase with high-energy electron beams. The electron beams displace an electron from the molecule and leave behind a...
8.4K
Mass Spectrometry of Amines01:15

Mass Spectrometry of Amines

In mass spectroscopy, amines undergo fragmentation to give parent ions with odd molecule weights. This observed mass spectrum follows the nitrogen rule; a molecule with an odd number of nitrogen atoms produces a molecular ion with an odd molecular weight. Amines undergo fragmentation through α cleavage, producing nitrogen-containing cations—iminium ions—and alkyl radicals. Mass spectra of aromatic and cyclic aliphatic amines exhibit strong molecular ion peaks, but acyclic...
5.3K