Probing the gas-phase folding kinetics of peptide ions by IR activated DR-ECD

Abstract

The effect of infrared (IR) irradiation on the electron capture dissociation (ECD) fragmentation pattern of peptide ions was investigated. IR heating increases the internal energy of the precursor ion, which often amplifies secondary fragmentation, resulting in the formation of w-type ions as well as other secondary fragments. Improved sequence coverage was observed with IR irradiation before ECD, likely due to the increased conformational heterogeneity upon IR heating, rather than faster breakdown of the initially formed product ion complex, as IR heating after ECD did not have similar effect. Although the ECD fragment ion yield of peptide ions does not typically increase with IR heating, in double resonance (DR) ECD experiments, fragment ion yield may be reduced by fast resonant ejection of the charge reduced molecular species, and becomes dependent on the folding state of the precursor ion. In this work, the fragment ion yield was monitored as a function of the delay between IR irradiation and the DR-ECD event to study the gas-phase folding kinetics of the peptide ions. Furthermore, the degree of intracomplex hydrogen transfer of the ECD fragment ion pair was used to probe the folding state of the precursor ion. Both methods gave similar refolding time constants of approximately 1.5 s(-1), revealing that gaseous peptide ions often refold in less than a second, much faster than their protein counterparts. It was also found from the IR-DR-ECD study that the intramolecular H. transfer rate can be an order of magnitude higher than that of the separation of the long-lived c/z product ion complexes, explaining the common observation of c. and z type ions in ECD experiments.

Figure 1

ECD spectra of substance P with 150 ms electron irradiation and (a) no IR irradiation; (b) 200 ms IR irradiation immediately before the ECD event; and (c) 200 ms IR irradiation immediately after the ECD event. * marks electronic noise.

Figure 2

ECD (upper) and DR-ECD (lower) spectra of fibrinopeptide B with 150 ms electron irradiation and (a) no IR irradiation; (b) 150 ms IR irradiation immediately before the ECD event; and (c) 150 ms IR irradiation, followed by a 400 ms delay, before the ECD event. Insets illustrate the pulse sequence used in each experiment. For clarity, only c, z and w type ions are labeled, and # indicates secondary fragments corresponding to additional side chain losses from z-type ions. Dashed cleavages are observed in AI-ECD only.

Figure 3

First order decays of the intact c₇/z₇ ion pair abundance as a function of ejection time when ECD of fibrinopeptide B was carried out at various delays after the IR irradiation. Solid lines are single exponential fits.

Figure 4

Percentage of the fibrinopeptide B ions that remained unfolded (as calculated from the DR-ECD experiment) at various time delays after the IR irradiation was turned off. Solid line is the single exponential fit.

Figure 5

(a) Expanded regions of the ECD spectra of fibrinopeptide B showing that the percentage of the hydrogen transfer product (z₇) increased as the delay between the IR and ECD events increased. (b) Percentage of the fibrinopeptide B ions that remained unfolded (as calculated from the ratio of z₇^• and z₇ abundances) at various time delays after the IR irradiation was turned off. Solid line is the single exponential fit.

Figure 6

Linear fit of the ratio of the z₇^• and z₇ abundances as a function of the ion separation rate constant as calculated from the DR-ECD experiments.