Electron Capture Dissociation in a Branched Radio-Frequency Ion Trap

Nov 25, 2014 - time-of-flight mass spectrometer using novel branched radio frequency ion trap architecture. With this device, a low-energy electron be...
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Electron Capture Dissociation in a Branched Radio-Frequency Ion Trap Takashi Baba,* J. Larry Campbell, J. C. Yves Le Blanc, James W. Hager, and Bruce A. Thomson AB Sciex, 71 Four Valley Drive, Concord, Ontario, L4K 4V8, Canada S Supporting Information *

ABSTRACT: We have developed a high-throughput electron capture dissociation (ECD) device coupled to a quadrupole time-of-flight mass spectrometer using novel branched radio frequency ion trap architecture. With this device, a low-energy electron beam can be injected orthogonally into the analytical ion beam with independent control of both the ion and electron beams. While ions and electrons can interact in a “flow-through” mode, we observed a large enhancement in ECD efficiency by introducing a short ion trapping period at the region of ion and electron beam intersection. This simultaneous trapping mode still provides up to five ECD spectra per second while operating in an informationdependent acquisition workflow. Coupled to liquid chromatography (LC), this LC-ECD workflow provides good sequence coverage for both trypsin and Lys C digests of bovine serum albumin, providing ECD spectra for doubly charged precursor ions with very good efficiency. modes: (1) flow-through or (2) simultaneous trapping of precursor ions with a continuous beam of electrons. Efficient ECD is a product of a unique RF ion guide (Figure 1a),21 whose branched structure allows the intersection of a lowenergy electron beam with the analytical ion beam of the mass spectrometer. Here, we demonstrate the use of this instrument for obtaining ECD spectra on peptides eluted from an ultrahigh pressure LC (UHPLC) with an ECD acquisition rate of 5 spectra/second. While traditional proteomics analyses generally utilize nanoLC, our use of UHPLC here is meant to demonstrate the compatibility of this system for the rapid characterization of protein- and peptide-based therapeutics in high-throughput environments in support of either discovery of development groups in the pharmaceutical industry. Using this challenging UHPLC-ECD-MS workflow, we obtained high sequence coverage for both the trypsin (75%) and Lys C (85%) digests of bovine serum albumin in this novel example of a simultaneous trapping mode ECD.

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lectron capture dissociation (ECD)1−12 and electron transfer dissociation (ETD)13−16 are two dissociation techniques used in mass spectrometry to complement traditional collisional induced dissociation (CID) data for peptide and protein analyses. These two related methods, commonly grouped using the abbreviation ExD (where “x” represents either “C” for “capture” or “T” for “transfer), dissociate peptide and protein ions with retention of labile post-translational modifications (e.g., phosphorylation,5 glycosylation,6,7 etc.), unlike CID. These characteristics allow ExD to be employed for top-down analyses2 and de novo sequencing3,4 of modified peptides and proteins. While ECD was initially available only with Fourier transform ion cyclotron resonance (FTICR) mass spectrometers,1 many other MS forms are now capable of performing either ECD or ETD.8−13 These ExD-capable devices can be categorized into two general forms: (1) those that trap analyte ions during the ExD stage1−10,12−17 and (2) those that perform the dissociation step as analyte ions are continuously flowing through the ExD region.11,18−20 Trapping analyte ions during the ExD step can be implemented on the time scale of liquid chromatography (LC) separation (i.e., 2−5 ExD events per second for tryptic peptide ions). However, the duty cycle is lower than for a flow-through mode because, while trapping one analyte ion population, the rest of the analytical ion beam goes unused. Conversely, a flow-through ExD configuration can allow ∼100% usage of the analyte ion beam, but reported ExD efficiencies (i.e., ExD product ion signal/total precursor ion signal) are typically quite low (≤1%).11 Here, we describe a novel ECD-enabled quadrupole time-offlight (Q-TOF) mass spectrometer that can operate in either © 2014 American Chemical Society

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EXPERIMENTAL SECTION Chemicals and Materials. HPLC-grade acetonitrile was obtained from Caledon Laboratory Chemicals (Georgetown, ON), and distilled deionized water (18 MΩ) was produced inhouse using a Millipore (Billerica, MA) Integral 10 water purification system. Bovine serum albumin (BSA), formic acid, neurotensin, dithiothrietol, and iodoacetamide were purchased Received: October 8, 2014 Accepted: November 25, 2014 Published: November 25, 2014 785

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Figure 1. (a) ECD cell. The two types of hatching indicate different phases of the applied RF voltage. (b) Six-way-crossed pseudopotential minimum by the RF field (dashed line) and dc lenses for axial confinement. (c) ECD cell device was installed between a quadrupole mass filter, Q1, and dissociation quadrupole, Q2.

from Sigma-Aldrich (Oakville, ON), while trypsin and endoprotease Lys C were purchased from Promega (Madison, WI). ESI tuning mix (Agilent Technologies, Mississauga, ON) was diluted 1/100 in neat acetonitrile. Sample Preparation. BSA was solubilized (1 mg/mL) and digested following protocols for trypsin24 and for Lys-C,25 including reduction and alkylation of any cystines. Once completed, the digested protein samples were aliquoted and frozen at −25 °C. Prior to analysis, the BSA digests were diluted to 2 pmol/μL in 100% water with 0.1% formic acid and pipetted into capped LC vials. Sample Introduction. For experiments involving the direct infusion of analytes (e.g., neurotensin, ESI tuning mix, etc.), solutions were infused into the ESI source at 3 μL/min using a syringe pump. The chromatographic separations were performed using a Prominence XR UHPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with two LC-20AD XR pumps, a temperature-controlled autosampler (SIL-20AC XR), and a column oven (CTO-20AC). Samples in the autosampler were held at 15 °C. An Aeris C-18 column (3.6 μm particle diameter, XB-C18, 100 Å pore size, 100 × 2.1 mm, Phenomenex, Torrance, CA) was used to separate the peptides within the digests; this column was held at 40 °C inside the column oven. The two pumps delivered a reverse-phase gradient (mobile phase A: H2O/0.1% formic acid; mobile phase B: ACN/0.1% formic acid) at a flow rate of 300 μL/min. Peptides were eluted from the column using a linear gradient of 2%−40% B over 8 min, followed by high organic (95% B) for 1 min, and a further 3.4 min of re-equilibration at 2% B. For each experiment, 30 pmol of BSA digest was injected on-column. Quadrupole Time-of-Flight Mass Spectrometer with ECD Cell. All experiments were conducted using a modified quadrupole time-of-flight (Q-TOF) mass spectrometer22 with an ECD cell installed between lens electrode IQ2 and quadrupole Q2 (Figure 1c). As such, this instrument can also be employed using conventional beam-type CID dissociation by accelerating precursor ions through the ECD cell and into Q2. A more specific discussion of the ECD cell’s design and operation (including additional components and their respective manufacturers) is given in the Results and Discussion section (vide infra). Precursor ions were generated by electrospray ionization (ESI) using a DuoSpray ion source (AB Sciex); the source parameters employed are listed in Table 1.

Table 1. Ion Source Parameters Employed during the LCECD-MS Experiments parameter

direct infusion

UHPLC

ion spray voltage source temperature curtain gas nebulizer gas desolvation gas

+5500 V 25 °C 10 psi 10 psi 0 psi

+5500 V 550 °C 25 psi 30 psi 70 psi

We assessed the efficiencies of the electron capture and the dissociation from “EC no D” precursors using the following formulas: [electron capture efficiency] = ( ∑ (ECD fragment ions) +

∑ (“EC no D”precursor ions))/( ∑ (ECD fragment ions)

+

∑ (“EC no D”precursor ions) + (residual precursor ions)) (1)

[dissociation efficiency] =

∑ (ECD fragment ions) ∑ (ECD fragment ions) + ∑ (“EC no D”precursor ions) (2)

where the “EC no D” ions are defined as charge-reduced precursor ions [M + nH]•(n−1)+ and [M + (n − 1)H](n−1)+ (M = mass of the neutral precursor peptide). Instrument Control and Data Analysis. The BSA digests were analyzed using an information-dependent acquisition (IDA) workflow as acquired using Analyst 1.6 TF software (AB Sciex). Instrument parameter control for the ECD mode (or ECD cell) was performed using a research-grade acquisition system developed in-house. The ECD data for the trypsin- and Lys-C-digested BSA samples were searched using Mascot (Matrix Science, Boston, MA, U.S.A.) using the following parameters: Swissprot database, Mammalia taxonomy, fixed carbamidomethyl modification on cysteine, peptide tolerance of 1.2 Da, MS/MS tolerance of 0.6 Da, peptide charges 2+, and FTMS-ECD as the instrument (as ECD is most commonly found on FTICRs).

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RESULTS AND DISCUSSION Design of the ECD Cell. The architecture of the ECD cell is based on a crossed quadrupole array21 that is a combination

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Figure 2. Operation voltage and scan functions for the ECD cell device. (a) dc potential configuration along the z-axis (ion path) and x-axis (electron path by blue line). (b) Scan function for trapping mode. (c) Scan function for a type of trapping mode used to measure Figure 5. (d) Scan function for a type of trapping mode used to measure Figure 6.

electrode (gate 1, Figure 1c). The bias of gate 1 was set low when the electron beam was stopped, while a second gate electrode (gate 2−incorporated into L4) was set low to repulse the electron beam toward the trap center. To avoid ion diffusion in along the y-axis of the ECD cell, we placed two electrodes (L5 and L6) on the top and bottom of the ECD quadrupole array. In addition, vanes were inserted on L5 and L6 along the z-axis to accumulate precursor ions along the xaxis of the array’s intersection. A magnetic field, B, is also applied along the x-axis within the ECD cell to minimize radial diffusion of the electron beam (Figure 1c). This magnetic field (0.17 T) is generated by two N42 grade ring neodymium magnets (RX8CC, K & J Magnetics Inc. PA) that are attached to the L3 and L4. The combination of the quadrupole’s electric fields and the magnetic field focuses electrons at the termini of the quadrupole array but weakly defocuses electrons at the array’s center depending on the phase of the RF field. Electron motions were modeled using SIMION (Scientific Instrument Services, Inc., NJ) and showed such defocusing in the inlet branch, but interestingly, the electrons were refocused in another branch (Figure S-1, Supporting Information). This focusing can be explained by the RF phase inversion (i.e., RF phase was inverted across the center of the ECD cell while electrons traversed this region, leading to beam defocusing). This focusing/defocusing of the electron beam allows multiple return trips for an electron beam between L3 and L4 without significant RF heating and current losses when the electron beam turns around at gate 2. As a result, RF voltages with higher frequencies and amplitudes can be applied to the ECD cell without significant electron heating,8,12 such that product ions with wider mass ranges can be trapped and ultimately detected. Since the ECD cell has been implemented using this branched configuration, the precursor ion control lenses (L1

of eight L-shaped electrodes (Figure 1a). There are four branches, each comprising a linear quadrupole with the trap diameter of 2r0 (=8 mm), to which radio frequency voltage (Vrf = 0−400 V(zero‑to‑peak)) with a frequency of Ω/2π was applied. The RF employed was an 802-kHz sinusoidal waveform generated by a function generator (33120A, Agilent Technologies, CA) and amplified by a homemade RF amplifier before applying a tank LC circuit. This frequency provided an optimal m/z range from ∼m/z 100 to at least m/z 3000 (the maximum presently examined) for trapping ions in the ECD cell, as well as optimal ECD efficiency. The ECD cell is designed to have precursor ions and electrons interact at the minimum pseudopotential23 of the quadrupole array, which lies at the junction of the crossed array (dashed lines in Figure 1b). Precursor ions that are isolated by quadrupole 1 (Q1) enter from the left-hand side of the diagram of Figure 1c, along the z-axis. Two dc lenses, L1 and L2, gate ion flow through the ECD cell; voltages applied to these lenses can establish either flow-through or ion trapping conditions within the cell (Figure 2a). Ions exit the cell along the z-axis to the right-hand side for extraction to the TOF mass spectrometer. The quadrupole used for electron beam injection is positioned along the x-axis of the array. Here, two lenses (L3 and L4) are positioned and biased positively relative to the ECD cell to confine ions along the x-axis. Both L3 and L4 also serve as magnetic pole pieces to lead the magnetic flux into the ECD cell (vide infra). These biases were controlled by software based on LabView (National Instruments Corporation, TX). DC voltage configuration is shown in Figure 2a. An electron beam can be injected from an emitter through a hole in L3 into the ECD cell, where it travels along the pseudopotential minimum such that RF heating of the electrons is minimized.8 The electron emitter (emt), a yttria (Y2O3) coated iridium disk (ES-525, Kimball Physics, NH), generates thermal electrons that are extracted by a gate 787

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Figure 3. Pure and trapping ECD spectra of triply protonated neurotensin. Accumulation time for each spectrum was 0.5 s. Opening duration of exit lens: L2 was changed from (a) 0 ms as pure flow through to (b) 10 ms to (c-1) 19 ms for trapping. (c-2) Shows the vertically expanded spectrum of (c-1). The product ions and residual precursor ions were extracted during the “open” period. (d) Plot of product ion intensities as a function of the trapping mode reaction periods.

and L2) function independently from the electron beam generation (unlike FTICR configurations). In effect, we are injecting precursor ions into a stable electron “gas” similar to a beam-type CID experiment in Q-TOF or triple quadrupole instruments where ions are injected into an inert collision gas. In the ECD cell, a pressure of ∼3 mTorr helium is employed for cooling of ion’s kinetic motion; in Q2, a similar pressure of nitrogen gas is employed for ion transmission and for CID (if applicable). For optimal ECD performance, a voltage offset of 5 V relative to Q0 (i.e., the “collision energy” setting) was applied to the ECD cell during collection of ECD spectra to avoid collisional dissociation of the precursor ions.. In addition, the DC bias applied to the electron emitter was set to −1 V relative to the ECD cell (i.e., electrons had 1 eV kinetic energy) in order to obtain optimal ECD efficiency. Characterization of the ECD Cell. To obtain optimal performance from this ECD cell, we characterized its ability to perform two key functions. The first attribute is the cell’s ability to trap precursor ions efficiently, which can influence the overall sensitivity of the ECD process by better trapping of both precursor ions and their respective ECD fragments. Figure S-2a, Supporting Information, shows ion trapping characteristics of the ECD cell as a function of RF amplitude (Vo‑p) for three different ions. In this evaluation, a trapping scan function (Figure 2b) was used (10 ms for ion loading, 20 ms for trapping, and 1 ms for extraction) with no electron beam. Both L1 to L4 were set at +15 V, while L5 and L6 were set at +6 V relative to the ECD cell. For each of the ions trapped in the cell, we observed a sharp onset of ion trapping (i.e., normalized intensity of the trapped ions) as a function of applied RF amplitude, with more gradual decrease in efficiency as the RF

amplitude was increased further. The RF amplitudes measured at the declining edge of trapping efficiency for each ion (e.g., ∼150 V0‑p for m/z 322) were matched to calculated low mass cut off (LMCO: Mathieu stability parameter: q = 0.908) in the branches with a linear quadrupole structure. The presence of trapped ions beyond these LMCO voltages suggest that ions were located at the center of the ECD cell where LMCO was not defined because of higher-order multipole RF fields. Ultimately, trapped ions were lost at very high RF amplitudes because the pseudo potential well at the cell’s center became steep so that the ions were pushed back toward the branches where the ions were unstable. In addition to the applied RF amplitude, we evaluated the influence of the L5 and L6 lens voltages on the ion trapping efficiency of the cell. Figure S-2b, Supporting Information, shows the measured trapping efficiencies for singly charged ions of m/z 322 for different RF amplitudes and dc biases on L5 and L6. As the lens bias was increased, more ions were trapped as the higher dc bias on the lenses pushed the trapped ions toward the center (intersection) of the ECD cell away from the distal (and unstable) regions along the z-axis. This suggests that ECD efficiency should be increased when the precursor ions located in the distal branches along the z-axis are pushed into the x-axis branches by the voltages applied to the vanes on the L5 and L6 (Figure 1b). The second key attribute we evaluated was the efficiency of the ECD process in this new cell by using triply protonated neurotensin [(M + 3H)3+] as the precursor ion. First, we evaluated a pure “flow-through” ECD experiment, wherein lens voltages were established to allow precursor ions to pass unabated through the cell. The electron beam was established 788

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Figure 4. Model calculation of simultaneous trapping ECD mode and conventional trapped ECD mode. (a) Residual (unreacted) precursor ion intensity and first ECD product intensity by simultaneous trapping mode (solid lines) and by conventional trapped ECD mode (dashed lines). (b) First ECD product intensity in simultaneous trapping ECD mode and trapped ECD mode for different precursor loading times from 2 to 10 ms.

during this passage through the cell such that the precursor ions and any ECD fragment ions would reside in the cell for only as long as transit through the cell required (∼μs). As shown in Figure 3a, no discernible ECD product ions were observed in this condition, which can be explained by the very short ionelectron interaction period in the cell (∼μs). By comparison, the required duration for an electron capture event in similar ECD devices is between 1 and 50 ms.4,7,8,12 To improve the ECD efficiency without adding a long trapping period, we applied a simultaneous trapping mode scan function (Figure 2c) where the L1 was kept open and L2 was made repulsive for a brief period of time to control the ECD duration. In addition, the electron beam remained on and was trapped in the ECD cell by the closed gate 2 (Figure 2a) during the period of precursor ion trapping. Precursor ions were injected into the ECD cell continuously (similar to the flow-through experiments) but the effect of establishing the L2 barrier for only a few milliseconds is dramatic (Figure 3b,c-1,d), as the intensity of ECD fragment ions improved greatly. These results can be rationalized using a simple model to describe this simultaneous trapping mode ECD event, where the electron beam continuously provides electrons to the precursor ion population. Some variables in this model include i0 as constant precursor ion flow per unit time from the ion source and k as electron capture rate of the precursor per unit time. (We will neglect secondary electron capture events here since the 3+ precursor ion should undergo a primary electron capture event at a higher rate than either a 2+ or 1+ charged ECD product.26) In our simultaneous trapping mode operation, ions and electrons interact simultaneously while the exit gate is closed to precursor ions. This differs from a more conventional trapping mode, where precursor ions are introduced for a duration of Δt, and then, electron beam is applied. In such a sequence, the number of the residual precursor ions (N(t)) and the total number of these ions undergoing a primary electron capture event (Np(t)) are given by N(t ) = i0t

and

Np(t ) = 0

N(t ) = i0Δt exp[−k(t − Δt )] Np(t ) = i0Δt − N(t)

for

for

t < Δt

However, in our trapping mode, ions and electrons are injected continuously but the exit gate is closed, creating the relationships: N(t ) = i0(1 − exp[ −kt ])/k

Np(t ) = i0t − N(t ) (5)

Figure 4a shows a comparison between the conventional trapping mode and the simultaneous trapping mode for a typical case where k−1= 5 [ms] and Δt = 5 [ms] and i0 is normalized to 1. The figure suggests the advantage of the simultaneous trapping mode operation compared to pure flow through operation. Because the product curve in the simultaneous trapping mode is quadratic, t = 0 ms represents a reaction time too short to provide any ECD fragment ions; the “flow-through” ECD experiment (Figure 3a) validates this point (i.e., t ∼ μs is insufficient). However, in the simultaneous trapping mode, when the L2 barrier is established for a period of ∼1/k or longer, efficient ECD fragmentation is achieved (Figure 3d). However, in the actual ECD experiments, the use of too long an ion injection time decreased the overall efficiency (data not shown), potentially because the spatial distribution of the precursor ions can be larger than the electron cloud. In addition, too long an ECD reaction period can result in secondary (or higher) electron capture events. For more heavily charged precursor ions (e.g., large proteins), this can result in more complicated ECD fragmentation patterns. Hence, this simple model suggests that our simultaneous trapping mode should be operated with t ∼1/k (i.e., 1−10 ms) for Z ≥ 3+ precursors and t ∼10−50 ms for Z = 2+. Figure S-3, Supporting Information, displays ECD spectra of substance P in the simultaneous trapping mode and the conventional trapping mode, demonstrating that the performance of this ECD cell matches that of other analogous techniques. Figure 4 displays another advantage of the simultaneous trapping mode over the conventional trapping mode. In the conventional trapping mode, the total number of primary ECD products is limited to the precursor ions introduced for Δt, even when the electron beam is subsequently applied for a very long period. In contrast, the simultaneous trapping ECD process provides linearly increasing amounts of product ions as the experiment’s duration is increased. As shown in Figure 4b, the simultaneous mode generates more products than the conventional mode in any ion loading duration.

(3)

and t > Δt

and

(4) 789

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Figure 5. Electron capture efficiency of different precursor charge states of (a) Z = 2+, (b) Z = 3+, and (c) Z = 4+. Dissociation efficiency of different precursor charge states of (d) Z = 2+, (e) Z = 3+, and (f) Z = 4+. Two types of BSA digests by trypsin and Lys C were injected into the LC-ECDTOF MS. Arrows in (a) and (d) indicate the peptide: VPQVSTPTLVEVSRSLGK, whose ECD spectrum is shown in Figure 6a.

Figure 6. Two ECD spectra for (a) 2+ precursor peptide ion, which is indicated by the arrows in Figure 5a,d, and (b) 5+ precursor peptide ions from BSA digest. Black, red, and green coloring represent 1+, 2+, and 3+ charged states of fragments.

LC-ECD Analyses of Protein Digests: Mapping Sequence Coverage and Charge-State ECD Efficiencies. Upon optimizing both the ion trapping capabilities and the ECD mode of operation, LC-ECD experiments were conducted to assess the sequencing capabilities of this novel ECD cell. Bovine serum albumin (BSA), digested by either trypsin or Lys C, was analyzed by LC-ECD, with a UHPLC used to introduce the peptides. This configuration was selected to demonstrate the speed at which this ECD cell could acquire sequence-quality ECD fragment ion spectra. The simultaneous trapping ECD mode (Figure 2d) was employed with some

minor adjustments. First, the electron beam was turned off during the product ion extraction period (∼1 ms) to avoid attracting these ions back toward the center of the ECD cell with the dense electron cloud. Second, the ECD cell’s entrance lens (L1) was also closed (i.e., set to +10 V) during the extraction period to avoid contamination of precursor ions that went to Q2 without exposure to the electron beam. The duration of the simultaneous trapping period was 49 ms for all charge states to enhance the intensity of the fragment ions, especially for doubly charged precursor ions. Prior to each ECD event, a survey TOF MS scan was acquired, and from these 790

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data, we employed IDA criteria to select only the five most intense precursor ions with charge states from Z = 2+ to 5+ for ECD fragmentation. The ECD spectra were accumulated for 200 ms with five ECD spectra acquired per second. The sequence coverage obtained for the BSA digests analyzed by LC-ECD were 85% (Lys C digest) and 75% (trypsin digest). These levels are quite acceptable given that the majority of precursor ions (for both trypsin and Lys C digests)27 are only doubly charged. These ions are especially challenging to dissociate by ETD methods and often require abandoning such activation methods in favor of the moretraditional CID.27 However, using this ECD cell, we obtained electron capture efficiencies from 15% to 75% and dissociation efficiencies from 25% to 100% for doubly charged precursor ions. Figure 5 displays both electron capture and dissociation efficiencies as a function of m/z and charge state for precursor ions identified by MASCOT as BSA peptides (trypsin and Lys C digest results combined here). Electron capture and dissociation efficiencies for precursor ions with higher charge states (Z ≥ 3+) were expectedly higher than for the 2+ precursor ions (Figure 5). For all ions, dissociation efficiencies decreased as the m/z of the precursor ions increased in spite of the use of a constant and high electron capture efficiency. This can be attributed to several factors, including the vibrational cooling of larger ions that abates fragmentation (i.e., more stable “EC no D” precursor ions), as well as lower overall charge density for peptides of different molecular weights but similar charge states (i.e., less effect of intra-ion Coulomb repulsion during dissociation).28 Electron capture efficiency was low for doubly charged precursor ions with high m/z as the worst case scenario, but still, this ECD technique worked well enough to annotate fragment species in database searching (Figure 6a). The results displayed in Figure 5 also reveal that dissociation efficiency is not necessarily an area in need of great improvement, although improving the electron capture rate for Z = 2+ precursor ions would be useful. Achieving this by increasing the electron beam’s intensity is unlikely, given that this feature has been saturated already due to electron space charge in the ECD cell (i.e., higher temperature for the electron emitter did not enhance the electron capture efficiency). However, increasing the strength of the ECD cell’s magnetic field beyond 0.17 T is another opportunity for improvement, as well as increasing the volume of overlap between the electron cloud and the precursor ions. Further opportunities for improved efficiencies include using beam-type collision of the “EC no D” product ions or charge reduced species (CRS•) to enhance the overall ECD efficiency.29

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +1 905 660 2623. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Pablo Dominguez for his excellent contributions towards the design of the ECD cell. (For Research Use Only. Not for use in diagnostic procedures.) The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX is being used under license Copyright 2014 AB SCIEX.

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CONCLUSIONS A new ECD cell device using a branched radio frequency ion trap was developed. This high-throughput ECD device housed in a Q-TOF MS obtained five ECD spectra per second using a simultaneous trapping mode of operation. With its short trapping period and dramatically enhanced electron capture, the simultaneous trapping ECD mode will be promising for proteomics because of this mode’s cycle time and the fact that it is easy to tune the independently controlled precursor ions and electron populations. While the simultaneous trapping mode should be beneficial for many applications, nothing prevents the use of a conventional trapping mode for fundamental studies of various electron−ion-based dissociation methods. 791

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dx.doi.org/10.1021/ac503773y | Anal. Chem. 2015, 87, 785−792