Identification and characterization of EX1 kinetics in H/D exchange mass spectrometry by peak width analysis

Abstract

Proteins that undergo cooperative unfolding events display EX1 kinetic signatures in hydrogen exchange mass spectra. The hallmark bimodal isotope pattern observed for EX1 kinetics is distinct from the binomial isotope pattern for uncorrelated exchange (EX2), the normal exchange regime for folded proteins. Detection and characterization of EX1 kinetics is simple when the cooperative unit is large enough that the isotopic envelopes in the bimodal pattern are resolved in the m/z scale but become complicated in cases where the unit is small or there is a mixture of EX1 and EX2 kinetics. Here we describe a data interpretation method involving peak width analysis that makes characterization of EX1 kinetics simple and rapid. The theoretical basis for EX1 and EX2 isotopic signatures and the effects each have on peak width are described. Modeling of EX2 widening and analysis of empirical data for proteins and peptides containing purely EX2 kinetics showed that the amount of widening attributable to stochastic forward-and back exchange in a typical experiment is small and can be quantified. Proteins and peptides with both obvious and less obvious EX1 kinetics were analyzed with the peak width method. Such analyses provide the half-life for the cooperative unfolding event and the relative number of residues involved. Automated analysis of peak width was performed with custom Excel macros and the DEX software package. Peak width analysis is robust, capable of automation, and provides quick interpretation of the key information contained in

A mechanistic study of the H/D exchange reactions of protonated arginine and arginine-containing Di- and tripeptides

“…Reaction efficiencies (k exp /k coll ) contained in column 1 of Table 1 are the average values obtained from multiple experiments. Relative reaction rates (k= 1 -k= 4 ; also listed in Table 1) for the product ions formed by exchange were obtained by numerically fitting the exact solutions of the differential kinetics equations (assuming pseudofirst-order approximations) to the ion temporal profiles. Fitting procedures are preferred over experimentally measured kinetics data owing to low confidence values in the measured rates for slow reactions, i.e., reactions having rate constants of less than 1 ϫ 10 Ϫ10 cm 3 molecule Ϫ1 s Ϫ1 .…”

“…Although H/D exchange reagents can access all labile hydrogen atoms of small peptides, the accessibility of exchange reagents toward labile hydrogen sites of larger peptides and proteins is conformation dependent [1], thus the rate of H/D exchange reactions and the number of hydrogen atoms exchanged can be used as a structural probe [2]. H/D exchange and mass spectrometry can be combined to study both gas-phase and solutionphase H/D exchange reactions [3][4][5][6][7][8][9], and the kinetics of H/D exchange can be utilized to infer the presence of distinct conformations of both solution-phase [10 -12] and gas-phase peptide and protein ions [13]. For example, McLuckey et al studied the gas-phase H/D exchange of bradykinin [M ϩ H] ϩ ions (amino acid sequence RPPGFSPFR) with DI and D 2 O, and they interpreted the observed bimodal distributions of product ions as evidence for two non-interconverting ion conformations having different reactivities toward deuterating reagents [14].…”

Abl N-Terminal Cap Stabilization of SH3 Domain Dynamics

“…NCap3 was more deuterated than SH3 ( Figure 3B, left) because it contained more residues. While apparent in the raw spectra, the change in peak width with time that is indicative of EX1 kinetics and partial unfolding is most obvious in a peak-width plot where an increase in width correlates with the unfolding event (22). The centroid of the peak in a peak-width plot is the approximate unfolding half-life.…”

Dynamics of the Tec‐family tyrosine kinase SH3 domains

“…In the most clear cases, the two isotope envelopes are fully resolved from each other, whereas in other cases they are not, depending on how many residues are involved in the EX1-driven event. 26 To analyze EX1 spectra where there are clearly resolved isotope envelopes, the intensity of the unfolded (higher mass) species is monitored with time to extract the rate constant and half-life (t 1/2 ) for unfolding. In the case of non-resolved isotope envelopes, the width of the total isotope distribution at half maximum (FWHM) is monitored as a function of exchange time and a peak-width plot 26 of these data provides the half-life of unfolding.…”