Evaluation and optimization of electron capture dissociation efficiency in fourier transform ion cyclotron resonance mass spectrometry

Abstract

Electron capture dissociation (ECD) efficiency has typically been lower than for other dissociation techniques. Here we characterize experimental factors that limit ECD and seek to improve its efficiency. Efficiency of precursor to product ion conversion was measured for a range of peptide (ϳ15% efficiency) and protein (ϳ33% efficiency) ions of differing sizes and charge states. Conversion of precursor ions to products depends on electron irradiation period and maximizes at ϳ5-30 ms. The optimal irradiation period scales inversely with charge state. We demonstrate that reflection of electrons through the ICR cell is more efficient and robust than a single pass, because electrons can cool to the optimal energy for capture, which allows for a wide range of initial electron energy. Further, efficient ECD with reflected electrons requires only a short (ϳ500 s) irradiation period followed by an appropriate delay for cooling and interaction. Reflection of the electron beam results in electrons trapped in or near the ICR cell and thus requires a brief (ϳ50 s) purge for successful mass spectral acquisition. Further electron irradiation of refractory precursor ions did not result in further dissociation. Possibly the ion cloud and electron beam are misaligned radially, or the electron beam diameter may be smaller than that of the ion cloud such that remaining precursor ions do not overlap with the electron beam. Several ion manipulation techniques and use of a large, movable dispenser cathode reduce the possibility that misalignment of the ion and electron beams limits ECD efficiency. [12], and histones [13][14][15]. Despite its proven analytical utility, ECD suffers from limited conversion efficiency of precursor to product ions. The dispenser cathode electron source has decreased the irradiation period and increased the reproducibility of ECD [16], but has not significantly improved the efficiency [17]. Factors such as charge neutralization and larger number of observed fragmentation pathways make ECD difficult to apply to large precursor ions of low abundance. In addition, it can be quite difficult to establish and maintain optimized ECD operating conditions. The emergence of FT-ICR instruments in biological mass spectrometry makes the robustness and efficiency of ECD even more critical.In this work, we systematically characterize experimental factors that limit ECD and seek to improve its efficiency. Research is designed to investigate all aspects of the ECD experiment, including duration of electron irradiation, electron energy and flux, multiple reflection of the electron beam versus a single pass through the ICR cell, ejection of trapped electrons from the ICR cell following the ECD event, and spatial overlap of the electron beam with the trapped ions. We report our conditions for optimized ECD and discuss their physical rationale and general implications.

Clustered O-Glycans of IgA1

“…We have previously demonstrated that AI-ECD of IgA1 HR distinguished between a single population and an isobaric mixture of differentially glycosylated HR peptides (20). However, fragmentation by use of ECD is less efficient than the more traditional collision-induced dissociation in the conversion of parent ions to fragments (ϳ30 versus Ͼ70% conversion) (40). As such, a small subpopulation of isobaric IgA1 HR O-glycoforms with sites of Gal deficiency as indicated by the N-terminal fragments would not be observed in the presence of a dominant single population.…”

Periodic sequence distribution of product ion abundances in electron capture dissociation of amphipathic peptides and proteins

“…Gated ion trapping was used without cooling gas. For ECD experiments, a variable delay period (typically 50 ms at 10 V trapping voltage) was used to optimize ion magnetron motion phase [41]; a 3-mm-diameter electron beam (1-100 ms) was then injected into the ICR trap, followed by an electron clean-up event (100 ms) [40,42]. The cathode potential during electron injection was Ϫ5 V and kept at ϩ10 V otherwise.…”

“…Accelerating grid voltage was at ϩ5 V during electron injection and at Ϫ200 V otherwise. A multiple-pass electron injection regime was used (transfer octopole DC offset Ϫ60 V) [40,42]. For AI-ECD experiments, precursor ions were activated by CO 2 IR laser irradiation (50 -100 ms at ϳ25 W continuous-wave laser output power; the laser was equipped with a 2.5ϫ beam expander to improve overlap with the ion cloud) before electron injection [39].…”

“…ECD FT-ICR MS of six doubly protonated horse myoglobin tryptic fragments ( [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16], [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31], [32][33][34][35][36][37][38][39][40][41][42] Figure S2) and with vibrational activation ( Figure 5) confirm preferential and sometimes periodic product ion formation from peptides with mainly ␣-helical or ␤-turn secondary structure in solution (before protein digestion). Predominantly z-ions are observed because of preferential charge retention at the C-terminal Lys or Arg basic residue upon electron capture, as expected for doubly charged tryptic peptides.…”

Characterization of phosphate-containing metabolites by calcium adduction and electron capture dissociation

“…The fragmentation efficiency in SORI-CAD is, as expected, higher (22% for NAD and 40% for NADP under the conditions used) than that in ECD (11% for NAD and 12% for NADP). Efficiencies were calculated based on the total product ion abundance divided by the initial precursor ion abundance (measured in a separate experiment), as proposed by McFarland et al [58].…”