Protein conformational stability probed by Fourier transform ion cyclotronresonance mass spectrometry

We have probed the conformational stability of cellular retinoic acid-bindi ng protein I, a predominantly beta-sheet protein, using hydrogen/deuterium (H/D) exchange in solution. Transiently populated intermediate states were detected using H/D exchange measurement under mildly denaturing conditions (pH 2.5 and room temperature). By inducing collisionally activated dissocia tion in the nozzle-skimmer region of the electrospray source of an FT ICR m ass spectrometer (MS), residue-specific information was obtained as to the degree of protection of backbone amide hydrogen atoms as a function of exch ange time. The measurements do not appear to be influenced by intramolecula r proton mobility in the gas phase. Multiply charged fragment ions covering half of the protein sequence were readily assigned using the extremely hig h resolution of FT ICR, allowing in some cases protection at individual ami de hydrogen atoms to be measured. The results reveal distinct structural re gions featuring very different backbone protection patterns. The high data acquisition rate of the FT ICR MS results in significant improvement of tem poral resolution over NMR spectroscopy.