FIGURE 3.

D/H-exchange kinetics in isotropic solution. Exhaustively deuterated peptides were assayed for back-exchange. (A) Exemplary mass spectra of the triply-charged syb-wt ion at different time points. The spectrum at t = 0 min was recorded after exchange under stop conditions (3 min incubation on ice at pH 2.5) where only very labile deuteriums exchange (see text for details). Low intensity isotopic envelopes at calculated masses ∼22 Da above the dominant envelopes likely originate from Na⁺-adducts. (B) A comparison of the exchange kinetics exhibited by syx, syb-wt, syb-multA, syb-L8, and L16. The data points at t = 0 correspond to the numbers of amide deuteriums seen after exchange under stop conditions. The data were fit with a three-term exponential function assuming D = 19 at t = 0 min (continuous lines; see Experimental Procedures for details). (C) Comparison of exchange rate constants that were calculated for deuterium classes whose size was equivalent for each peptide. No k_A value syx is given for syx, whose k_A is at least as high as that of syb but could not be precisely calculated due to improper curve fitting at the earliest time points. All values represent means ± SD of at least three independent measurements.