Effects of charge state on fragmentation pathways, dynamics, and activation energies of ubiquitin ions measured by blackbody infrared radiative dissociation

Abstract

Blackbody infrared radiative dissociation spectra of the (M + 5H)5+ through (M + 11H)11+ ions of the protein ubiquitin (8.6 kDa) formed by electrospray ionization were measured in a Fourier-transform mass spectrometer. The 5+ ion dissociates exclusively by loss of water and/or ammonia, whereas the 11+ charge state dissociates only by formation of complementary y and b ions. These two processes are competitive for intermediate charge state ions, with the formation of y and b ions increasingly favored for the higher charge states. The y and b ions are formed by cleavage of the backbone amide bond on the C-terminal side of acidic residues exclusively, with cleavage adjacent to aspartic acid favored. Thermal unimolecular dissociation rate constants for the dissociation of each of these charge states were measured. From the temperature dependence of these rates, Arrhenius activation parameters in the rapid energy exchange limit are obtained. The activation energies (Ea) and preexponential factors (A) for the 5+, 8+, and 9+ ions are 1.2 eV and 10(12) s-1, respectively. These values for the 6+ and 7+ ions are 0.9-1.0 eV and 10(9) s-1, and those for the 10+ and 11+ ions are 1.6 eV and 10(16)-10(17) s-1. Thus, with the exception of the 5+ ion, the higher charge states of ubiquitin have larger dissociation activation energies than the lower charge states. The different A factors observed for production of y and b ions from different precursor charge states indicate that they are formed by different mechanisms, ranging from relatively complex rearrangements to direct bond cleavages. These results clearly demonstrate that the relative dissociation rates of large biomolecule ions by themselves are not necessarily a reliable indicator of their relative dissociation energies, even when similar fragment ions are formed.

Figure 1

Blackbody infrared radiative dissociation spectra of the 6+ to 11+ charge states of ubiquitin (a–f, respectively) taken from the kinetic data. Temperature and reaction delay for each spectrum are (a) 6+, 145 °C, 90 s; (b) 7+, 170 °C, 140 s; (c) 8+, 161 °C, 240 s; (d) 9 +, 151 °C, 300 s; (e) 10+, 151 °C, 200 s (expansion of molecular ion at 156 °C with no reaction delay inset); and (f) 11+, 151 °C, 60 s.

Figure 2

Data for the dissociation of ubiquitin (a) 7+ and (b) 11+ charge states fit to first-order kinetics at the temperatures indicated.

Figure 3

Arrhenius plots for dissociation of the 5+ to 11+ charge states of ubiquitin over a temperature range of 134–202 °C.

Figure 4

Simplified reaction diagram for formation of complementary y and b ions from ubiquitin showing possible reaction pathways consistent with Arrhenius A factors of 10⁹–10¹⁷ s⁻¹.

Scheme 1