Design and Performance of an ESI Interface for Selective External Ion

and external ion accumulation has been shown to increase both sensitivity and analysis duty cycle. Instrument ver- satility is further improved by ion...
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Anal. Chem. 2001, 73, 253-261

Design and Performance of an ESI Interface for Selective External Ion Accumulation Coupled to a Fourier Transform Ion Cyclotron Mass Spectrometer Mikhail E. Belov, Evgenii N. Nikolaev,† Gordon A. Anderson, Harold R. Udseth, Thomas P. Conrads, Timothy D. Veenstra, Christophe D. Masselon, Mikhail V. Gorshkov,‡ and Richard D. Smith*

Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352

The coupling of Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) with electrospray ionization has advanced the analysis of large biopolymers and provided the basis for high-throughput protein characterization (e.g., for rapid “proteome” analyses). In this work, the combination of high-performance capillary liquid chromatography with FTICR mass spectrometry and external ion accumulation has been shown to increase both sensitivity and analysis duty cycle. Instrument versatility is further improved by ion preselection followed by ion accumulation in an external linear quadrupole ion trap. The interface was tested with a 3.5-T FTICR mass spectrometer and evaluated with a number of peptides and proteins whose molecular weights ranged from 500 to 66 000. A significant increase in the sensitivity, duty cycle, and dynamic range over that of the previously used accumulated trapping was achieved, exhibiting a detection limit of ∼10 zmol (∼6000 molecules) for smaller proteins such as cytochrome c. Capillary LC external accumulation interface with FTICR was successfully applied for the study of whole-proteome mouse tryptic digests. Recent works have demonstrated the extensive potential of mass spectrometry in conjunction with separation techniques, such as high-performance liquid chromatography (HPLC) and capillary isoelectric focusing (CIEF), for proteomic studies.1-4 Due to the vast volume and complexity of information generated, e.g., by tryptic digests of complex mixtures of proteins, such studies pose enormous challenges for all aspects of mass spectrometry performance and subsequent data analysis. Coupling electrospray ionization to Fourier transform ion cyclotron resonance mass † Visiting scientist from the Institute of Energy Problems of Chemical Physics, Russian Academy of Sciences, 117829 Moscow, Russia. ‡ Permanent address: The Institute of Energy Problems of Chemical Physics, Russian Academy of Sciences, 117829 Moscow, Russia. (1) Yates, J. R. 3rd. J. Mass Spectrom. 1998, 33, 1-19. (2) Jensen, P. K.; Pasˇa-Tolic´, L.; Anderson, G. A.; Horner, J. A.; Lipton, M. S.; Bruce, J. E.; Smith, R. D. Anal. Chem. 1999, 71, 2076-2084. (3) Li, J.; Kelly, J. F.; Chernushevich, I.; Harrison, D. J.; Thibault, P. Anal. Chem. 2000, 72, 599-609. (4) Veenstra, T. D.; Martinovic, S.; Anderson, G. A.; Pasˇa-Tolic´, L., Smith R. D. J. Am. Soc. Mass Spectrom. 2000, 11, 78-82.

10.1021/ac000633w CCC: $20.00 Published on Web 12/16/2000

© 2001 American Chemical Society

spectrometry (FTICR MS) has demonstrated a mass resolution of ∼8 000 000 for smaller proteins,5 a sensitivity in the attomoleto-zeptomole range,6-9 and mass accuracies of better than 2 ppm.10 Key to further increases in sensitivity are issues related to the efficiency of ion utilization. A significant increase in duty cycle has been demonstrated using ion accumulation in an external octopole trap followed by gated trapping.11 As ion accumulation and detection are spatially separated, the next packet of ions can be accumulated while the previous packet is being analyzed, providing a duty cycle that can approach 100%. Other improvements derived from external accumulation are reported to be enhanced signal-to-noise ratio and mass resolving power.11 Optimal sensitivity requires not only a high accumulation duty cycle but also a high trapping efficiency (as well as trapped ion populations that do not exceed certain sizes). The efficiency of trapping based on ion-neutral collisions is directly proportional to the number density of neutral particles or to the pressure in the FTICR cell. The utility of pressures above 10-5 Torr in an FTICR cell is ultimately determined by the speed of the pumping arrangement used to achieve the ultrahigh vacuum. (Higher pressures used, for example, in extended LC-FTICR analyses will also result in faster saturation of cryopumps or the cryopanels used in our instrumentation.) Therefore, accumulation of ions in an external linear ion trap at a higher pressure should increase the trapping efficiency compared with accumulated trapping in the FTICR cell.12 (5) Shi, S. D.; Hendrickson, C. L.; Marshall, A. G. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11532-11537. (6) Valaskovic, G. A.; Kelleher, N. L.; Little, D. P.; Aaserud, D. J.; McLaferty F. W. Anal. Chem. 1995, 67, 3802-3805. (7) Valaskovic, G. A.; Kelleher, N. L.; McLafferty, F. W. Science 1996, 273, 1199-1201. (8) Belov, M. E.; Gorshkov, M. V.; Udseth, H. R.; Anderson, G. A.; Tolmachev, A. V.; Prior, D. C.; Harkewicz, R.; Smith, R. D. J. Am Soc. Mass Spectrom. 2000, 11, 19-23. (9) Belov, M. E.; Gorshkov, M. V.; Anderson, G. A.; Udseth, H. R.; Smith, R. D. Anal. Chem. 2000, 72, 2271-2279. (10) Bruce, J. E.; Anderson, G. A.; Wen, J.; Harkewicz, R.; Smith, R. D. Anal. Chem. 1999, 71, 2595-2599. (11) Senko, M. W.; Hendrickson, C. L.; Emmett, M. R.; Shi, S. D.-H.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1997, 8, 970-976. (12) Beu, S. C.; Laude, D. A., Jr. Int. J. Mass Spectrom. Ion Processes 1991, 104, 109-127.

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To realize the benefits of external trapping, efficient transfer from the external trap to the FTICR trap is required. To achieve close to 100% efficiency of gated trapping in the FTICR, the temporal profile of an ion pulse ejected from the external ion trap should match the period of ion axial oscillation in the FTICR trap. When combined, the external ion accumulation and the efficient gated trapping should result in higher overall instrumental sensitivity. The dynamic range and achievable sensitivity in many applications of an FTICR mass spectrometer equipped with an external accumulation interface will generally also be substantially increased by introducing ion selection prior to ion accumulation in the external trap. This approach has several advantages over ion selection in the FTICR cell based on stored waveform inverse Fourier transform (SWIFT)13 or on resonant ion ejection,14 due to more limited charge capacity of the FTICR cell. To avoid saturating the FTICR cell, higher abundance species should be filtered out either externally or during ion accumulation in the FTICR cell. “Colored” noise waveforms and quadrupole excitation have been previously employed during lower resolution selective ion accumulation in the FTICR cell to significantly increase the dynamic range with FTICR.15 Ion selection at higher resolution should provide ejection of higher abundance ions prior to external accumulation and, as a result, allow “filling” of the FTICR cell with the species of interest. SWIFT or resonant ion ejection can then be applied to trim the ion population and to potentially broaden the dynamic range. In this work, we report on the development and implementation of selective external ion accumulation for FTICR mass spectrometry. An electrospray ionization (ESI)-external accumulation interface comprising three quadrupoles was modeled, designed, and implemented with a 3.5-T FTICR mass spectrometer. The interface was evaluated using peptides and proteins with molecular weights ranging from 500 to 66 000. A considerable increase in the duty cycle, dynamic range, and sensitivity was achieved compared with conventional accumulated trapping. Further, the instrumental performance of the mass spectrometer was evaluated in conjunction with on-line capillary LC using a complex polypeptide mixture from tryptic digestion of a mouse proteome extract. EXPERIMENTAL SECTION The FTICR mass spectrometer used in the present studies is based on a 3.5-T unshielded solenoid magnet (Oxford Instruments) and a vacuum system design previously described.8,9 The mass spectrometer incorporates the ESI ion source with an electrodynamic ion funnel8 and a quadrupole for collisional focusing, the external accumulation interface, an electrostatic ion guide, and a cylindrical dual cell combination. An Odyssey data station controlled the timing and potential distribution during the experiment. The vacuum system comprises six stages of differential pumping that gradually decrease the pressure from atmospheric to ultrahigh vacuum in the region of the FTICR trap. The ESI source and the external accumulation interface consist of four differentially pumped stages. (13) Wang, T.-C. L.; Ricca, T. L.; Marshall, A. G. Anal. Chem. 1986, 29352938. (14) Guan, S.; Marshall, A. G. Anal. Chem. 1993, 65, 1288-1294. (15) Bruce, J. E.; Anderson, G. A.; Smith, R. D. Anal. Chem. 1996, 68, 534541.

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ESI emitter voltages of ∼2 kV were applied to produce positive ions. The 0.75-mm-i.d., resistively heated stainless steel inlet capillary used for droplet desolvation was maintained at ∼120 °C and typically biased at +200 to +300 V. The first stage of differential pumping, which incorporates the electrodynamic ion funnel,8,9 was evacuated by a 43 L/s roots pump (Leybold AG, Cologne, Germany) to ∼1-3 Torr. The ions transmitted through the funnel exit electrode to the next stage were collisionally focused by a 150-mm-long quadrupole ion guide (9.525-mm rod diameter) operated in rf-only mode (typically 250 Vp-p at 430 kHz) at a pressure of 0.23 Torr (obtained using a 18 L/s mechanical pump, Leybold AG). The dc offset potentials of +15 to +20 V were typically applied to the collisional quadrupole rods to assist transmission of the ions through the 2-mm-i.d. conductance limit between the collisional quadrupole and external accumulation interface. The conductance limit was biased at +10 to +15 V. The external accumulation interface incorporating three quadrupoles (field radii of 4.1 mm, 9.525-mm rod diameter) is shown schematically in Figure 1. The assembly designed for collisional focusing, ion selection, and ion accumulation was fit in a 8-in.-o.d., six-way vacuum cross (MDC, Hayward, CA) and operated at a pressure of 5 × 10-5 Torr. Positioned at the interface input, a 150-mm-long quadrupole ion guide operated in rf-only mode at an amplitude of 560 Vp-p and a frequency of 2.1 MHz supplied to the +11-V dcbiased quadrupole rods by a commercial rf drive (Extranuclear Labs, Pittsburgh, PA). The middle, 100-mm-long, analytical quality quadrupole of the assembly was used for ion selection (referred to as the “selection quadrupole”). This quadrupole was enclosed in a differentially pumped vacuum can that allowed for pressure adjustment in the range of 10-2-10-4 Torr without affecting the base pressure in the rest of the interface. The rods and the 2-mmi.d. input and output conductance limits of the selection quadrupole were biased to +8, +6, and -19 V, respectively. The selection quadrupole was driven by an in-house-built high-Q head controlled by a function generator (model HP 33120A, Hewlett-Packard, Loveland, CO) and rf amplifier (model 100A150A, Amplifier Research, Souderton, PA). Two modes of ion selection have been used: (1) an rf/dc linear ramp and (2) rf-only selection based on resonant dipolar excitation.17 To implement the latter approach, two opposite rods of the selection quadrupole were coupled to the secondary coil of an auxiliary 1:1 transformer. The middle point of the transformer secondary coil was driven by the main rf drive at amplitudes of 300-1200 Vp-p and frequencies ranging from 500 to 700 kHz. The excitation waveform was then applied to the primary coil of the transformer. The 10-cm-long exit quadrupole of the interface used for ion accumulation (referred to as “accumulation quadrupole”) was segmented to provide an axial electric field for prompt ion ejection. An in-house-built high-Q head drove the accumulation quadrupole at a rf-amplitude of 220 Vp-p and a frequency of 525 kHz. A linear electric field gradient of ∼0.3 V/cm was applied along the accumulation quadrupole to create an axial potential well at the quadrupole exit. The rf voltage applied to the quadrupole segments (each rod was segmented (16) Gorshkov, M. V.; Pasˇa-Tolic´, L.; Udseth, H. R.; Anderson, G. A.; Huang, B. M.; Bruce, J. E.; Prior, D. C.; Hofstadler, S. A.; Tang, L.; Chen, L.-Z.; Willett, J. A.; Rockwood, A. L.; Sherman, M. S.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1998, 9, 692-700. (17) Campbell, J. M.; Collings, B. A.; Douglas, D. J. Rapid Commun. Mass Spectrom. 1998, 12, 1463-1474.

Figure 1. Schematic diagram of the interface for selective ion accumulation coupled to the ESI source. ESI source elements: (1) ion funnel, (2) collisional quadrupole, and (3) conductance limit between the ion guiding and the collisional quadrupoles. Elements of the interface: (4) ion guiding quadrupole, (5) entry plate of the selection quadrupole, (6) selection quadrupole, (7) exit plate of the selection quadrupole, (8) entry plate of the accumulation quadrupole, (9) accumulation quadrupole, and (10) exit plate of the accumulation quadrupole.

into 22 parts to maximize axial electric field penetration inside the segments) was de-coupled from dc and pulsed voltages generated using a in-house-built high-voltage multichannel amplifier through a RC circuit. The dc offset potentials of +12 and -2 V were applied to the 4-mm-i.d. input and output conductance limits of the accumulation quadrupole. To increase the instrument duty cycle, the ion guide quadrupole was also used for ion trapping during ion ejection from the accumulation quadrupole. The measurements of the pulsed total ion current (TIC) at the rear trapping plate of the FTICR cell were performed using a current-to-voltage converter with a conversion ratio of 1 V to 1 nA. It was found that the rise time of the RC circuit of the FTICR cell lengthened the front and tail of TIC waveforms by the up to 200 and 1000 µs, respectively. In a separate set of experiments the performance of the external accumulation interface was compared with that of a single longer quadrupole segment. The triple quadrupole interface was replaced by a 330-mm-long quadrupole 8,9 where the ions were trapped. Use of an external ion trap increases the effective ion emittance compared with continuous ESI source operation. Thus, the previously described electrostatic ion guide in the high-vacuum region of the mass spectrometer9,16 was modified. An additional Einzel lens was designed and positioned downstream of the acceleration region. A pressure of 7 × 10-10 was maintained in the FTICR ion cell region. The proteins and peptides were dissolved in a water/methanol/ acetic acid solution (49:49:2 v%) at different concentrations ranging from 2 pg/mL to 0.1 mg/mL (the lowest concentration was ∼10-12 M for horse cytochrome c). The solutions were infused into the ESI source at a flow rate of 300 nL/min using a syringe pump (Harvard, South Natick, MA). HPLC/FTICR MS data sets were obtained using a commercial HPLC system (Isco Inc., Lincoln, NE). Two identical samples of

B16 mouse cytosolic proteins were labeled with either a light or heavy isotopic version of biotinyliodoacetamidyl 4,7,10 trioxatridecanediamine.18 The tryptic peptides were injected onto a 150 µm i.d. × 60 cm long capillary column packed with a 5-µm-diameter C18 separation medium (PoroS 20R2, Perspective Biosystem, Framingham, MA). A solvent gradient was used to elute the peptides using 0.4% acetic acid in water (solvent A) and 0.4% acetic acid in 80% acetonitrile (solvent B). The peptides were eluted using a linear gradient of 0-80% solvent B over 60 min. Solvents were delivered to the capillary column at a pressure of 6000 psi using two Isco model 100 DM pumps controlled by an Isco series D controller and a LC Packings Accurate microflow processor splitter (LC Packings, San Francisco, CA) resulting in a capillary flow rate of 1 µL/min. RESULTS AND DISCUSSION 1. Ion Accumulation in the Segmented Quadrupole. Sensitivity is probably the most important factor for many biological applications of mass spectrometry. The sensitivity of a FTICR mass spectrometer with an external ion trap depends on the efficiency of ion trapping in the external trap, transmission efficiency between the external trap and the FTICR cell, and efficiency of trapping in the FTICR cell. In this work, we have designed, implemented, and demonstrated instrumental approaches and modifications that enable significant advances in sensitivity. To maximize the efficiency of external trapping, the potential applied to the front trapping plate of a linear quadrupole trap should match the kinetic energy of the incoming ion beam. The potential applied to the rear trapping plate should ideally exceed the kinetic energy per charge of the fastest ions. The ions entering the trap would then need only a few collisions with a background (18) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechol. 1999, 17, 994-999.

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Figure 2. (A) Measured ion current for different accumulation times in the segmented quadrupole for an ESI of 10-5 M solution of horse myoglobin. (B) Intensity of TIC signals and the total ion charge detected at the rear trapping plate of the FTICR cell as functions of the accumulation time in the external accumulation quadrupole.

gas to be axially trapped. As soon these initial ions are captured in the 2-D quadrupole trap, the trapping efficiency is significantly enhanced by ion-ion collisions and becomes essentially nonlinear. Figure 2A shows the dependence of the signal intensity for horse myoglobin ions (∼10-5 M) detected at the rear trapping plate on the flight time for different accumulation times, i.e., the time interval when the rf field is applied to the collisional quadrupole. The area of each waveform is proportional to the total charge transferred to the FTICR cell for the indicated particular accumulation time. No axial field was applied to the accumulation quadrupole, and the ejection time was chosen to be long enough so that the quadrupole was completely emptied. Figure 2B shows the total charge transferred to the FTICR cell and the intensity of the TIC pulse as functions of the accumulation time. Both the integrated total charge and the signal intensity become saturated at accumulation times longer than 200 ms, which implies the capacity of the linear quadrupole trap has been exceeded. The maximum number of charges trapped in the accumulation quadrupole was ∼108. An important contribution to the time-of-flight (TOF) distribution of the ions ejected from the external accumulation quadrupole is the quadrupole purging time. To measure the purging time, the trapping potential applied to the exit plate of the accumulation quadrupole was dropped to -2 V so that trapped ions could exit the quadrupole during a short ejection event and then raised back to the level corresponding to the optimum trapping conditions. The duration of the ion ejection event was varied while the TIC pulse was monitored at the rear plate of the FTICR cell. After a delay of 3-5 ms, the above trapping potential was reduced again to -2 V to completely purge the ions remaining in the accumulation quadrupole. The purging time was determined based on the ratio of the signal intensities for the two ejections. Figure 3A,B shows typical TIC signal at the rear trapping plate and the ratio of TIC intensities in two ejections. The time for complete purging of the accumulation quadrupole with an axial field of 0.2 V/cm was ∼500 µs. 256

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The TOF distributions of horse myoglobin charge states are shown in Figure 3C,D. The TOF distributions of externally accumulated ions were measured by trapping the ions in the FTICR cell at different delay times between the ejection pulse and the cell gating pulse. To minimize measurement errors, the potentials at the front and rear trapping plates of the cell were raised simultaneously as an ion cloud transited the cell. The full peak widths at half-maximum peak height (fwhm) of both distributions were less than 300 µs at an ejection time of 400 µs. It is noteworthy that the fwhm of the TOF distributions was consistent with the quadrupole purging time. As an ion cloud ejected from the accumulation quadrupole expands in all dimensions due to Coulombic repulsion, the fwhm of the TOF distributions indicates that time-lag focusing similar to the delayed extraction in TOF mass spectrometers19,20 was achieved during ion ejection. The period of ion axial oscillation in the cylindrical cell can be estimated as follows:21

Taxial ) 2πxma2/2zeVtrβ

(1)

where a is the length of the FTICR cell, m is the ion mass, β ) 2.84 is the geometrical factor of the cylindrical cell, Vtr is the trapping voltage, z is the charge state, and e is the elementary charge. For the [M + 17H]17+ ion of horse myoglobin Taxial ∼ 300 µs at Vtr ) 10 V. Hence, the efficiency of gated trapping should approach 100% at a delay time corresponding to the maximum of TOF distributions. (19) Wiley: W. C.; McLaren. I. H. Rev. Sci. Instrum. 1955, 26, 1950-1954. (20) Spengler, B.; Cotter, R. J. Anal. Chem. 1990, 62, 793-796. (21) Kofel, P.; Allemann, M.; Kellerhals, H.; Wanczek, K.-P. Int. J. Mass Spectrom. Ion Processes 1990, 98, 1-24.

Figure 3. (A) TIC pulse measured for a solution of horse myoglobin (∼10-5 M) at the rear trapping plate of the FTICR cell (2) and the trigger pulse (1). The first TIC pulse corresponds to a 300-µs-long ejection after a 50-ms-long accumulation of horse myoglobin ions from a 10-5 M solution. The second pulse is due to the ion population remaining in the accumulation quadrupole after the first ejection. The axial electric field in the accumulation quadrupole is 0.2 V/cm. (B) TIC pulse intensities at different ejection times: ([) intensity of the first signal peak in (A), (0) second peak intensity, and (2) intensity of the first signal peak as a percentage of the total TIC. (C) Time-of-flight distributions of horse myoglobin (∼10-5 M) charge states [M + 14H]14+ and (D) [M + 16H]16+ after 400-µs-long ejection under the conditions of no axial field present and a field gradient of 0.3 V/cm in the accumulation quadrupole. The ions were accumulated for 20 ms.

Comparison of the TOF distributions for myoglobin ions ejected from the accumulation quadrupole operating at different axial electric fields indicated that lower charge states at a given m/z are discriminated against when no axial electric field is present in the quadrupole. The integrated total charge trapped in the FTICR cell after ion ejection from the axial field-free accumulation quadrupole was measured to be ∼80% of that after ion ejection from the quadrupole when an axial field gradient of 0.2 V/cm was used. The difference is mainly due to the signal intensities of lower charge states (see Figure 3D). Ion ejection from the axial fieldfree quadrupole is determined by the space-charge repulsion, with higher charge states having higher mobility in the dense ion cloud and leaving the quadrupole at shorter ejection times. The 400-µs ejection time was found to be insufficient to efficiently purge the lower charge state myoglobin ions from such a quadrupole. Further increase in the axial field gradient resulted in signal decrease, possibly because the higher electric field modulates the ion’s kinetic energy, increasing it above the transmission “energy window” of the electrostatic ion guide. A practical tradeoff between the TOF distributions temporal profiles and the FTICR signal intensities was found at an axial electric field gradient of 0.3 V/cm; i.e., increasing the electric field gradient above 0.3 V/cm narrowed TOF distributions but decreased the FTICR signal intensities due to lower transmission efficiency. At 400-µs ion ejection time and an axial electric field gradient of 0.3 V/cm, the efficiency of ion ejection from the accumulation quadrupole was close 100%. It should be mentioned that due to limited ion energy modulation no apparent time-of-flight separation was observed for m/z ranging 500-1500, consistent with our modeling. Since the

instrument was designed to be used in conjunction with capillary LC, by adjusting the potentials during ion ejection from the accumulation quadrupole, we deliberately reduced the time-offlight separation to minimize time-of-flight discrimination against ions in the m/z range of 500-1500. After trapping the ions in the FTICR cell, the ion translational energy needs to be reduced to allow the use of lower trapping potentials, which yielded higher mass resolution. For an ion population close to the charge capacity of the FTICR cell, one would expect ion-ion interactions to provide effective cooling process in the time scale of a few hundred milliseconds.11 We studied the signal intensities of [M + 16H]16+ horse myoglobin and [M + H]+ bradykinin ions as a function of the cooling time with no gas present in the cell. For both the protein and the peptide, the signals reach the plateau at a cooling time of ∼50 s, which makes cooling based on ion-ion interactions generally ineffective for LC/MS experiments. However, soft-gas assistance (i.e., injection of a small amount of nitrogen gas), which increases the pressure in the FTICR by less than 1 order of magnitude (6 × 10-9 Torr), results in effective ion cooling in less than 1 s. Such a sustained gas load is easily tolerated by our cryopanel system. 2. Ion Selection. After the external ion accumulation in the segmented quadrupole and ion transfer to the FTICR cell were optimized, ion selection prior to accumulation was implemented. Two different regimes of ion selection such as a rf/dc linear ramp and rf-only resonant dipolar excitation were employed. The theory and practice of ion m/z selection based on rf/dc mass filtering has been extensively developed.22-24 The basic approach for achieving higher mass resolution is to choose the Analytical Chemistry, Vol. 73, No. 2, January 15, 2001

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scan line, which intersects with the ion stability diagram at the point of apex. The apex is characterized by Mathieu’s parameter q of 0.7.

q ) 4zeVrf/mω02r02

(2)

where Vrf is the peak-to-ground rf amplitude, z is the ion charge state, e is the elementary charge, m is the ion mass, ω0 is the rf-field angular frequency, and r0 is the quadrupole inscribed radius. On the basis of eq 2, rf amplitudes for selection of specific m/z ions were chosen. The dc voltage, U, corresponding to the apex, is governed by the relationship

U ) 0.169Vrf

(3)

Due to imperfections of the quadrupolar electric field caused by the circular cross section of the rods, fringing fields at the quadrupole entrance, exit, etc., the rf and dc voltages predicted from eqs 2 and 3 were found to deviate from those experimentally determined by less than 10%. The mass spectra of a mixture of the five peptides, leucine enkephalin, bradykinin, gramicidin S, angiotensin I, and substance P, at concentrations of 6 × 10-6, 1.7 × 10-5, 1.6 × 10-5, 2.3 × 10-5, and 1.2 × 10-5 M, respectively, are presented in Figure 4. The singly charged peptide ions were selected with respect to their m/z by the selection quadrupole and then accumulated for 80 ms in the segmented quadrupole. The sodiated ions of angiotensin I and the protonated species of substance P were selectively accumulated at a mass resolution of ∼100 (Figure 4D,E). To investigate the dynamic range of selective accumulation, the selection quadrupole was tuned up to eject all the peptide ions but leucine enkephalin. Figure 4 shows the mass spectra of the five-peptide mixture with no selectivity during accumulation for 80 ms (A) and the selective accumulation of leucine enkephalin for 80 (B) and 500 ms (C). More than 1 order of magnitude enhancement in the dynamic range results from the 6-fold increase in the accumulation time. When complex protein digests (e.g., in proteomic studies) are being analyzed, it would be highly beneficial to selectively and simultaneously remove higher abundance m/z peaks dispersed across the entire mass spectrum in order to avoid saturating the FTICR cell capacity. As rf/dc mass filtering is limited by one m/z region of the mass spectrum at a time, rf-only resonant dipolar excitation appears to be a complementary approach. When compared with rf-only quadrupolar excitation, whose spectrum of resonant frequencies can be complex with considerable structure,25 the dipolar excitation spectrum is rather simplified. In the first region of ion stability diagram at q < 0.7, the ion motion can be presented as superposition of rapid oscillations and a smooth drift in a harmonic pseudopotential well.26,27 The latter (22) Paul, W.; Steinwedel H. Z. Naturforsch. 1953, 8a, 448-452. (23) Paul, W.; Raether, M. Z. Phys. 1955, 140, 262-265. (24) March, R. E.; Hughes, R. J. In Quadrupole storage mass spectrometry: in Chemical Analysis; Winefordner, J. D., Ed.; J. Wiley & Sons: London, 1991, pp 31-52. (25) Sudakov, M.; Konenkov, N.; Douglas, D. J.; Glebova, T. J. Am. Soc. Mass Spectrom. 2000, 11, 10-18. (26) Dehmelt, H. G. Adv. Atom. Mol. Phys. 1967, 3, 53-72. (27) Gerlich, D. Adv. Chem. Phys. 1992, LXXXII, 1-176.

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Figure 4. Broadband (A) and selective (B-E) accumulation of a mixture of leucine enkephalin ([Leu + H]+), bradykinin ([Br + H]+), gramicidin S ([GrS + H]+), angiotensin I ([AnI + H]+), and substance P ([SubP + ]+) at concentrations of 6 × 10-6, 1.7 × 10-5, 1.6 × 10-5, 2.3 × 10-5, and 1.2 × 10-5 M, respectively. The selection quadrupole was operated in the rf/dc mass filtering mode. The peak-to-peak rf amplitude and dc voltages applied were as follows: (B) selective accumulation of [Leu + H]+ (389 Vp-p, +42 V, -24 V), accumulation time is 80 ms, average of 10 scans; (C) selective accumulation of [Leu + H]+, accumulation time is 500 ms, average of 10 scans; (D) selective accumulation of [AnI + H]+(1081 Vp-p, +101 V, -82.9 V) accumulation time is 80 ms, average of 10 scans; (E) selective accumulation of [SubP + H]+ (1120 Vp-p, +104 V, -86 V), accumulation time is 500 ms, average of 10 scans.

motion of a specific m/z is characterized by a unique fundamental resonant frequency, Ω, which can be estimated as

Ω ) (q/x8)ω0

(4)

The rf-only dipolar excitation was implemented with a continuous ion beam passing through the selection quadrupole. We found that operation of the selection quadrupole at q > 0.4 gave rise to a higher mass resolution during selection. An rf amplitude, Vrf, was chosen to correspond to q ∼ 0.7 for the lightest species of the selected m/z range. We studied resonant dipolar excitation with a mixture of bradykinin, gramicidin S, and substance P concentrated to 1.7 × 10-5, 8.5 × 10-6, and 2 × 10-6 M, respectively. The singly protonated ions for two peptides were resonantly ejected during one scan by synthesizing28 and applying a superposition of sine waveforms to the pair of opposite rods of the selection quadrupole. The waveform frequencies were determined by eq 4. The complete ejection of two peptides, without affecting the transmission of the third constituent of the mixture, indicates the applicability of this approach for the analysis of more complex samples. Mass spectra of horse myoglobin (∼10-6 M) for broadband accumulation and selective accumulation of all the charge states except for [M + 16H]16+ and [M + 14H]14+ ions are shown in Figure 5A,B. The [M + 16H]16+ and [M + 14H]14+ ions were ejected by applying a superposition of two sine waveforms at frequencies of 78.5 and 91 kHz and an amplitude of 3 V. Intensities of the other charge states were undisturbed, (28) Anderson, G. A.; Bruce, J. E. ICR-2ls, Pacific Northwest National Laboratory,1998.

Figure 5. Mass spectra of horse myoglobin (∼10-6 M). (A) Broadband accumulation for 20 ms. (B) Selective accumulation of all the charge states but [M + 16H]16+ and [M + 14H]14+ ions for 20 ms. [M + 16H]16+ and [M + 14H]14+ ions were resonantly ejected from the selection quadrupole by applying a superposition of two sine waveforms at frequencies of 78.5 kHz/91 kHz and a peak-to-peak amplitude of 3 V. (C) Mass spectrum of [M + 9H]9+ ion of cytochrome c at a concentration of 10-12 M. At a flow rate of 300 nL/min and an accumulation time of 5 s, the amount of sample consumed per one accumulation event was 20 zmol (∼12 000 molecules). The nonideal isotopic envelope evident for cytochrome c is attributed to the statistical limitations resulting from the relatively small number of [M + 9H]9+ ions actually trapped and analyzed.

indicating a minimal mass resolution of at least 20 was achieved during rf-only selective accumulation of the protein. The mass resolution of the rf-only selection was studied as a function of a number of parameters such as q, ion residence time, and pressure in the quadrupole, drive frequency, and the highest mass resolution was attained at q ∼ 0.6. When applied to bradykinin, the intensity of singly protonated ions was reduced by more than 1 order of magnitude without infringing the sodiated species, thus implying a mass resolution of ∼50. 3. Detection Limit. The detection limit of the FTICR mass spectrometer equipped with the external accumulation interface was tested with a number of peptide and protein samples. As an example, the mass spectrum of horse cytochrome c diluted to a concentration of 10-12 M is illustrated in Figure 5C. The solution was electrosprayed at a flow rate of 300 nL/min, and [M + 9H]9+ ions were selectively accumulated for 5 s in the segmented quadrupole. On the basis of sample consumption, the detection limit was found to be ∼10 zmol (∼6000 molecules). 4. Nonselective Accumulation in Longer rf-Only Quadrupole. The selective external accumulation in the external accumulation interface was compared with nonselective accumulation in the 330-mm-long rf-only quadrupole, which was repositioned to the 8-in.-o.d. six-way vacuum cross instead of the interface in Figure 1. Figure 6A shows the signal intensity from horse myoglobin ions (10-5 M), which was monitored at the rear trapping plate of the FTICR cell while sequentially ejecting the ions from the quadrupole after 100 ms-long accumulation. After each 300-µs-long ejection step, the potential at the quadrupole exit plate was raised to the level that corresponded to the optimum trapping conditions. As a result, the quadrupole was completely

purged for ∼2 ms. By integrating the waveforms, the ratios of the total charge purged during one particular ejection to the total charge accumulated, and also to that remaining in the quadrupole, were established. The dependences of the above ratios on the ejection number are shown in Figure 6B. Interestingly, in the absence of the axial field inside the quadrupole, the percent of total charge ejected to that remaining in the quadrupole was nearly independent of the ejection number, i.e., independent of ion population. This finding seems to be consistent with the mechanism of ion ejection based on space-charge repulsion, when the number of ions ejected is proportional to the number of ions left over in the quadrupole. As eject time should match the period of ion axial oscillation in the FTICR cell, the sequential ejection process was examined for the longer quadrupole. The delay between two neighboring ejects was chosen based on the ion cooling time in the FTICR cell (typically 1-2 s with soft gas assistance). After cooling the ion translational energy by a few electonvolts, the potential at the front trapping plate was decreased to allow for the next packet to enter the cell. The intensity of the monoisotopic peak of the singly protonated ion of bradykinin as a function of the transfer step number is presented in Figure 6C,D. The signal intensity revealed linear dependence on the ejection number up to 6 and was found to saturate upon increasing the number of ejects. The saturation is believed to be related to the charge capacity of the FTICR cell. Though higher efficiency of ion transfer from the longer quadrupole to the FTICR cell was achieved by sequential ejection, the duration of the total sequence increased by a factor of 3 compared with that of the accumulation interface. Therefore, the external accumulation in the longer rfonly quadrupole was found to be less suitable for HPLC/FTICR MS experiments than the selective external accumulation in the segmented quadrupole. 5. Capillary HPLC/FTICR MS. It is noteworthy that the selective external accumulation is characterized by a duty cycle approaching 100% compared with the lower duty cycle of the accumulated trapping in the FTICR cell (typically ∼10-2 but as much ∼10-1 in our systems incorporating cryopanels that allow higher pressure gas pulses). This is achieved by continuously transporting the ions through the collisional quadrupole to the external accumulation interface throughout the entire sequence, while trapping the ions in the accumulation quadrupole. During ion ejection from the accumulation quadrupole, the ions delivered from the ESI source are trapped in the ion guiding quadrupole of the assembly. The ion transfer from the ion guide quadrupole to the accumulation quadrupole was very efficient. The time required for cooling ion translational energy in the FTICR cell is then used to accumulate the next packet of ions. On-line capillary LC/FTICR MS (with 256K data sets) studies based on this strategy have been conducted. Figure 7 shows a reconstructed TIC chromatogram from the HPLC separation of the tryptic digest for a mouse proteome sample. On the basis of the signal intensity of the externally trapped ions, the optimal duration of one scan was 5 s. For a typical elution time of a chromatographic peak in the present work of about 20-25 s, four to five mass spectra were typically acquired per chromatographic peak. A total of 31 479 isotopic distributions were detected in single HPLC/FTICR MS experiment with 13 496 determined to be unique to 10 ppm mass measurement accuracy. From these data Analytical Chemistry, Vol. 73, No. 2, January 15, 2001

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Figure 6. (A) TIC pulse detected at the rear trapping plate of the FTICR cell (1) as a result of ion ejection from the 330-mm-long rf-only quadrupole. The trigger pulse (2) is shown for reference. B) The ratios of the total charge ejected to that accumulated (2) and remaining (1) in the quadrupole. C) The intensity of the monoisotopic peak of singly protonated ion of bradykinin as a function of the transfer step number. Ion accumulation was performed in the 330-mm- long rf-only quadrupole for 100 ms. D) Portions of the mass spectra corresponding to the first and sixth transfer steps.

Figure 7. The TIC chromatogram of the HPLC/FTICR MS data sets of the tryptic digest of a mouse proteome. The insets represent particular regions of the chromatogram and reveal the presence of light and heavy isotopic versions labeled with biotinyliodoacetamidyl-4,7,10trioxatridecanediamine.

(to be presented in detail elsewhere), labeled with either normal or heavy (D8) isotopic versions of biotinyliodoacetamidyl-4,7,10trioxatridecanediamine, 787 peptide pairs having mass difference of 8.06 Da were detected. These data will be used to identify cysteine-containing peptides by comparing the experimentally determined mass against a database of mouse tryptic peptides 260

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constructed from available genomic data utilizing the inherent cysteine constraint in each peptide. The ability to observe large numbers of peptides from a capillary LC separation provides an enabling capability for proteome research and represents a major breakthrough in high-throughput proteome analysis by FTICR MS.

CONCLUSIONS An interface for both nonselective and selective external ion accumulation comprising ion guiding, selection, and accumulation quadrupoles was developed, incorporated with an ESI 3.5-T FTICR mass spectrometer, and evaluated for a number of peptides and proteins. By segmenting the accumulation quadrupole, a prompt ion ejection step, that matched the period of ion axial oscillation in the FTICR cell was realized. Under optimized conditions of ion accumulation and ejection, the efficiency of gated trapping was estimated to be close to unity. To further expand the dynamic range of the instrument, ion selection prior to ion accumulation was implemented. Two ion selections using both rf/dc mass filtering and rf-only resonant dipolar excitation were employed and a mass resolutions of >100 and ∼50 were achieved, respectively, under conditions that did not sacrifice achievable sensitivity. Due to increased trapping efficiency, as compared with that of the accumulated trapping, the detection limit of ∼10 zmol (∼ 600 molecules) was attained. Finally, we have presented preliminary results for the ESI-FTICR mass spectrometer coupled with reversed-phase capillary LC and applied to the analysis of a tryptic digest of mouse proteins and where the duty cycle approached 100%. At a mass accuracy of better than 10 ppm, more than 13 496 unique isotopic distributions (i.e., putative peptides) were detected due to the enhanced sensitivity, dynamic range, and duty cycle of the instrument. The implementation of selective external

accumulation with ESI-FTICR instrumentation and its use with on-line CE or LC separations offers high potential for application to new proteomic approaches based upon the global proteolysis and characterization of the resulting highly complex peptide mixture. ACKNOWLEDGMENT The authors are grateful to Drs. Richard Harkewicz, Ljiljana Pasˇa-Tolic´, Kenneth Auberry, and Jeon Futrell, as well as Thomas Bailey (University of Delaware) for helpful discussions. We also thank Prof. R. Aebersold and co-workers (University of Washington) for their early help with use of the stable-isotope methodology. Dr. Gorshkov also thanks Russian Basic Sciences Foundation for support under Grant 99-04-49261. Parts of this research were supported by the NIH National Center fir Research Resources (RR12365) and the Office of Biological and Environmental Research, U.S. Department of Energy. Pacific Northwest National Laboratory (PNNL) is a multiprogram national laboratory operated by Battelle Memorial Institute for the U.S. Department of Energy under Contract DE-AC06-76RLO 1830.

Received for review June 12, 2000. Accepted November 3, 2000. AC000633W

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