biostudies-literatureFlint JAGSummer Science Undergraduate Research Fellowships ProgramGregory S. Call Academic Intern ProgramNational Science Foundatione70045https://www.ebi.ac.uk/biostudies/studies/S-EPMC11761709biostudies-literatureUnknown34(2)Hydrogen exchange mass spectrometry (HXMS) is a powerful tool to understand protein folding pathways and energetics. However, HXMS experiments to date have used exchange conditions termed EX1 or EX2 which limit the information that can be gained compared to the more general EXX exchange regime. If EXX behavior could be understood and analyzed, a single HXMS timecourse on an intact protein could fully map its folding landscape without requiring denaturation. To address this challenge, we developed a numerical simulation method called NumSimEX that models EXX exchange for arbitrarily complex folding pathways. NumSimEx fits protein folding dynamics to experimental HXMS data by iteratively comparing the simulated and experimental timecourses, allowing for determination of both kinetic and therProtein science : a publication of the Protein SocietyNumSimEX: A method using EXX hydrogen exchange mass spectrometry to map the energetics of protein folding landscapes.PMC117617091129152Strahan JJaswal SSHirsch ANi TPoterba TSong NFlint JAGCartagena AJSohl JLHan IWitten JDamjanovic JWagaman ASfalseNumSimEX: A method using EXX hydrogen exchange mass spectrometry to map the energetics of protein folding landscapes.Hydrogen exchange mass spectrometry (HXMS) is a powerful tool to understand protein folding pathways and energetics. However, HXMS experiments to date have used exchange conditions termed EX1 or EX2 which limit the information that can be gained compared to the more general EXX exchange regime. If EXX behavior could be understood and analyzed, a single HXMS timecourse on an intact protein could fully map its folding landscape without requiring denaturation. To address this challenge, we developed a numerical simulation method called NumSimEX that models EXX exchange for arbitrarily complex folding pathways. NumSimEx fits protein folding dynamics to experimental HXMS data by iteratively comparing the simulated and experimental timecourses, allowing for determination of both kinetic and ther2025-01-01T00:00:00Z2025 Feb2026-06-02T12:07:21.937Z2025-04-03T23:23:49.808ZS-EPMC117617093986538610.1002/pro.70045