Activated Ion Electron Capture Dissociation (AI ECD) of proteins: synchronization of infrared and electron irradiation with ion magnetron motion

Abstract

Here, we show that to perform activated ion electron capture dissociation (AI-ECD) in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer equipped with a CO(2) laser, it is necessary to synchronize both infrared irradiation and electron capture dissociation with ion magnetron motion. This requirement is essential for instruments in which the infrared laser is angled off-axis, such as the Thermo Finnigan LTQ FT. Generally, the electron irradiation time required for proteins is much shorter (ms) than that required for peptides (tens of ms), and the modulation of ECD, AI ECD, and infrared multiphoton dissociation (IRMPD) with ion magnetron motion is more pronounced. We have optimized AI ECD for ubiquitin, cytochrome c, and myoglobin; however the results can be extended to other proteins. We demonstrate that pre-ECD and post-ECD activation are physically different and display different kinetics. We also demonstrate how, by use of appropriate AI ECD time sequences and normalization, the kinetics of protein gas-phase refolding can be deconvoluted from the diffusion of the ion cloud and measured on the time scale longer than the period of ion magnetron motion.

Graphical abstract

Figure 1

Fragmentation efficiency for the 2+ ion of Substance P with (squares) different durations of ECD; (circles) different ECD time delays with ECD duration 70 ms; and (triangles) different IRMPD delays for IRMPD duration 20 ms.

Figure 2

Schematic representation of the geometrical arrangement for electron and IR laser beams in the ICR cell of Thermo-Finnigan LTQ FT. Top: overlap between ion trajectories, electron beam and IR beam in the plane A-A' at the entrance to the ICR cell.

Figure 3

Modulation imposed by ion magnetron motion on (a) relative intensities of (M + 6H)^5+• reduced ions produced by electron irradiation of ubiquitin (M + 6H)⁶⁺ ion, (b) depletion of the (M + 6H)⁶⁺ ion abundance by IRMPD, (c) post-ECD IR activation: depletion of (M + 6H)^5+• reduced ions.

Figure 4

Number of fragments from IR-AI ECD of ubiquitin cations versus laser fluency for (a) and (b) 85 ms IR activation followed by 10 ms ECD of the 6+ and 7+ ions, respectively, and (c) and (d) 10 ms ECD followed by 100 ms IR activation of the 6+ and 7+ ions, respectively. Squares represent the number of c' and z• ions, triangles for c• and z' ions, and circles for b and y ions.

Figure 5

Fragmentation diagrams for (a) ECD and (b)–(e) AI ECD of ubiquitin (M + 6H)⁶⁺ ion. (b) and (c) IR activation before ECD, ECD delay 85 ms, IR duration 85 and 25 ms, respectively; (d) 100 ms IR activation immediately after ECD; (e) IR activation before ECD, ECD delay 1165 ms, IR duration 85 ms.

Figure 6

Long-time evaluation of the efficiencies of electron capture, IRMPD and IR-AI ECD for ubiquitin ions. (a) (Squares) intensity of (M + 6H)^5+• reduced ion produced by electron capture from (M + 6H)⁶⁺ ion versus ECD delay; (a) (circles) IRMPD depletion of ubiquitin (M + 6H)⁶⁺ ion versus IRMPD delay; (b) (filled squares) pre-ECD IR activation: depletion of (M + 6H)^5+• reduced ion versus ECD delay; (b) (filled circles) pre-ECD IR activation: depletion of (M + 6H)^4+•• reduced ion versus ECD delay; (b) (hollow squares) post-ECD IR activation: depletion of (M + 6H)^5+• reduced ion versus IR activation delay; (b) (hollow circles) post-ECD IR activation: depletion of (M + 6H)^4+•• reduced ion versus IR activation delay.