Ion Mobility Spectrometer with Radial Collisional Focusing - American

Nov 24, 2004 - Ontario, Canada M3J 1P3, and MDS Sciex, 71 Four Valley Drive, Concord, Ontario, Canada L4K 4V8. An ion mobility spectrometer that has i...
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Anal. Chem. 2005, 77, 266-275

Ion Mobility Spectrometer with Radial Collisional Focusing Yuzhu Guo,† Jiaxi Wang,†,‡ Gholamreza Javahery,§,| Bruce A. Thomson,§ and K. W. Michael Siu*,†

Department of Chemistry and Centre for Research in Mass Spectrometry, York University, 4700 Keele Street, Ontario, Canada M3J 1P3, and MDS Sciex, 71 Four Valley Drive, Concord, Ontario, Canada L4K 4V8

An ion mobility spectrometer that has its mobility cell as a 20-segment quadrupole and functionally the q2 of a triple-quadrupole mass spectrometer has been assembled and tested. The combination of high cell pressure (maximum of 4 Torr of helium) and low axial field (20-160 V per 20.2 cm) results in negligible internal excitation of the ions despite applications of rf and axial fields. The presence of collisional focusing ensures efficient ion transmission and good sensitivity. Collision cross sections of atomic, cluster, peptide, and protein ions were measured and found comparable to literature and calculated cross sections. Mobility, K, of an ion is a measure of its effective size by virtue of its collision cross section, ΩT, with a given buffer gas, typically helium, under the influence of a weak electric field. Provided that the number density of the buffer gas, N, is sufficiently high and the imposed electric field, E, is sufficiently low, the drift velocity, vd, is proportional to E with K being the proportionality constant.

vd ) KE

(1)

ΩT can be determined from K using the Mason-Schamp equation:1,2

ΩT )

3ze 2π (16N )(µkT) (K1 ) 1/2

(2)

where z is the numerical charge, e is the electronic charge, µ is the reduced mass of the ion and helium, k is the Boltzmann constant, and T is the temperature. The collision cross section of an ion is one of the most readily measurable, albeit somewhat crude, structure-related parameters that have come under close scrutiny in the past decade or so. Coupled to comparisons with theoretical cross sections calculated * To whom correspondence should be addressed. Tel: (416)650-8021. Fax: (416)736-5936. E-mail: [email protected]. † York University. ‡ Current address: Regional Centre for Mass Spectrometry, McMaster University, Hamilton, ON, Canada. § MDS Sciex. | Current address: Ionics Mass Spectrometry Group, Inc., 130 Bradwick Drive, Unit 8, Concord, ON, Canada L4K 1K8. (1) Mason, E. A.; McDaniel, E. W. Transport Properties of Ions in Gases; John Wiley & Sons: New York, 1988. (2) Revercomb, H. E.; Mason, E. A. Anal. Chem. 1975, 47, 970-983.

266 Analytical Chemistry, Vol. 77, No. 1, January 1, 2005

from probable structures found by optimizations using molecular dynamics and first-principle methods, measurements of ΩT have allowed insights into three-dimensional structures of peptides and proteins in the gas phase.3-5 The first ion mobility spectrometers were designed for measuring the drift velocities and diffusion coefficients of small ions (1-3 atoms).6 The application of ion mobility spectrometry (IMS) to measurement of large ions from nonvolatile analytes, e.g., carbon clusters and biomacroions, introduced via electrospray ionization (ESI), laser vaporization, or matrix-assisted laser desorption/ionization (MALDI) opened up an entirely new vista in the evolution of IMS. This era of application to large ions began arguably with the pioneering work of Dole and co-workers,7 published in 1984, on characterization of lysozyme and polystyrene ions. In a series of papers that began in 1990, the Bowers’ group4,8-10 advanced the instrumentation and broadened the scope of applications in IMS/mass spectrometry (MS). Also in the early 1990s, Smith and co-workers11,12 reported ion mobility spectra of proteins in support of their MS work. Hill and co-workers13-15 demonstrated the capabilities of IMS detection for ESI of proteins and carried out the first full-scale investigation on cytochrome c and Triton X-100. The Jarrold group3,16-19 conducted a string of (3) Clemmer, D. E.; Hudgins, R. R.; Jarrold, M. F. J. Am. Chem. Soc. 1995, 117, 10141-10142. (4) Wyttenbach, T.; von Helden, G.; Bowers, M. T. J. Am. Chem. Soc. 1996, 118, 8355-8364. (5) Hoaglund-Hyzer, C. S.; Counterman, A. E.; Clemmer, D. E. Chem. Rev. 1999, 99, 3037-3079. (6) McDaniel, E. W.; Mason, E. A. The Mobility and Diffusion of Ions in Gases; John Wiley & Sons: New York, 1973; pp. 51-84 and references therein. (7) Gieniec, J.; Mack, L. L.; Nakamae, K.; Gupta, C.; Dole, M. Biomed. Mass Spectrom. 1984, 11, 259-268. (8) Kemper, P. R.; Bowers, M. T. J. Am. Soc. Mass Spectrom. 1990, 1, 197207. (9) von Helden, G.; Gotts, N. G.; Bowers, M. T. Nature 1993, 363, 60-63. (10) Bowers, M. T.; Kemper, P. R.; von Helden, G.; van Koppen, P. A. M. Science 1993, 260, 1446-1451. (11) Smith, R. D.; Loo, J. A.; Ogorzalek Loo, R. R.; Busman, M.; Udseth, H. R. Mass Spectrom. Rev. 1991, 10, 359-452. (12) Smith, R. D.; Loo, J. A.; Ogorzalek Loo, R. R. Udseth, H. R. Mass Spectrom. Rev. 1992, 11, 434-443. (13) Wittmer, D.; Chen, Y. H.; Luckenbill. B. K.; Hill, H. H., Jr. Anal. Chem. 1994, 66, 2348-2355. (14) Wu, C.; Siems, W. F.; Asbury, G. R.; Hill, H. H., Jr. Anal. Chem. 1998, 70, 4929-4938. (15) Wu, C.; Siems, W. F.; Klasmeier, J.; Hill, H. H., Jr. Anal. Chem. 2000, 72, 391-395. (16) Jarrold, M. F. J. Phys. Chem. 1995, 99, 11-21. (17) Shelimov, K. B.; Clemmer, D. E.; Hudgins, R. R.; Jarrold, M. F. J. Am. Chem. Soc. 1997, 119, 2240-2248. (18) Clemmer, D. E.; Jarrold, M. F. J. Mass Spectrom. 1997, 32, 577-592. 10.1021/ac048974n CCC: $30.25

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seminal work on the mobilities and structures of atomic clusters and proteins and introduced novel instrumental designs in IMS. The loss of protein ions’ energy as they traverse q2 of a triplequadrupole mass spectrometer was used by Douglas and coworkers20-23 as a measure of the collision cross section of the protein ions. Guevremont et al.24 reported the first interfacing of IMS to time-of-flight (TOF) MS. The advantages of orthogonal TOF MS detection for IMS were exploited by Clemmer and coworkers,25-28 who developed IMS TOF MS into a powerful analytical technique for protein analysis. Russell and co-workers29,30 interfaced MALDI IMS to orthogonal TOF MS, while Creaser et al.31,32 developed tandem ion trap/IMS. A high-field asymmetric waveform ion mobility spectrometer (FAIMS) was developed by Guevremont and co-workers.33-35 FAIMS separates ions by differences in the mobilities of ions at high electric field relative to those at low field, using a high-frequency periodic and asymmetric waveform. In 1997, Javahery and Thomson36 reported the first IMS results obtained using a prototype triple-quadrupole mass spectrometer fitted with a q2 which had been segmented axially into 10 pieces and across which an axial field could be imposed to effect mobility measurements. The radial rf field focused and confined ions, and minimized diffusional losses. Collision cross sections of protein ions in nitrogen and argon were reported. Unfortunately, the low pressure in q2 (a few mTorr) meant that the experimental conditions (E/N ) 38-1150 Td) lay outside of the low-field limit (see later). Here we report the construction of and the first results obtained on an ion mobility spectrometer/triple-quadrupole mass spectrometer whose IMS cell is a 20-segment quadrupole that functions as the q2 of the instrument. This 20-segment q2/IMS cell can be operated at a pressure of up to 4 Torr of helium. We will report that the presence of the rf field, in combination with high pressure, in the segmented quadrupole/IMS cell ensures efficient ion transmission via radial collisional focusing and minimal ion excitation by the rf field. Typical mobility measure(19) Jarrold, M. F. Acc. Chem. Res. 1999, 32, 360-367. (20) Covey, T.; Douglas, D. J. J. Am. Soc. Mass Spectrom. 1993, 4, 616-623. (21) Douglas, D. J. J. Am. Soc. Mass Spectrom. 1994, 5, 17-18. (22) Collings, B. A.; Douglas, D. J. J. Am. Chem. Soc. 1996, 118, 4488-4489. (23) Chen, Y.-L.; Collings, B. A.; Douglas, D. J. J. Am. Soc. Mass Spectrom. 1997, 8, 681-687. (24) Guevremont, R.; Siu, K. W. M.; Wang, J.; Ding, L. Anal. Chem. 1997, 69, 3959-3965. (25) Valentine, S. J.; Counterman, A. E.; Clemmer, D. E. J. Am. Soc. Mass Spectrom. 1999, 10, 1188-1211. (26) Srebalus, C. A.; Li, J.; Marshall, W. S.; Clemmer, D. E. Anal. Chem. 1999, 71, 3918-3927. (27) Hoaglund-Hyzer, C. S.; Clemmer, D. E. Anal. Chem. 2001, 73, 177-184. (28) Valentine, S. J.; Kulchania, M.; Srebalus Barnes, C. A.; Clemmer, D. E. Int. J. Mass Spectrom. 2001, 212, 97-109. (29) Gillig, K. J.; Ruotolo, B.; Stone, E. G.; Russell, D. H.; Fuhrer, K.; Gonin, M.; Schultz, A. J. Anal. Chem. 2000, 72, 3965-3971. (30) Ruotolo, B. T.; Gillig, K. J.; Stone, E. G.; Russell, D. H.; Fuhrer, K.; Gonin, M.; Schultz, J. A. Int. J. Mass Spectrom. 2002, 219, 253-267. (31) Creaser, C. S.; Benyessar, M.; Griffiths, J. R.; Stygall, J. W. Anal. Chem. 2000, 72, 2724-2729. (32) Colgrave, M. L.; Bramwell, C. J.; Creaser, C. S. Int. J. Mass Spectrom. 2003, 229, 209-216. (33) Purves, R. W.; Guevremont, R.; Day, S.; Pipich, C. W.; Matyjaszczyk, M. S. Rev. Sci. Instrum. 1998, 69, 4094-4105. (34) Purves, R. W.; Guevremont, R.; Day, S.; Pipich, C. W.; Matyjaszczyk, M. S. Rev. Sci. Instrum. 1999, 70, 1370-1383. (35) Guevremont, R.; Purves, R. W. J. Am. Soc. Mass Spectrom. 1999, 10, 492501. (36) Javahery, G.; Thomson, B. J. Am. Soc. Mass Spectrom. 1997, 8, 697-702.

ments were conducted with an axial field of 20-160 V per 20.2 cm, giving much lower E/N values of 3.06-24.4 Td and resulting in negligible collisional heating. Measured collision cross sections of atomic and protein ions will be reported and compared with literature and calculated collision cross sections. EXPERIMENTAL SECTION Instrumentation. The ion mobility spectrometer with radial collisional focusing was built from ion optic and electronic components of the commercially available API 3000 triplequadrupole mass spectrometer (MDS Sciex). Custom components were machined or assembled at York University and MDS Sciex. The IMS cell is based on Javahery and Thomson’s design that integrates components of a drift cell and segmented quadrupole.36 Functionally, the 20-segment quadrupole/IMS cell served as q2 of the triple-quadrupole mass spectrometer. To enable high operating pressure (4 Torr of helium, maximum) in the IMS cell, while maintaining sufficiently low operating pressure elsewhere, the segmented quadrupole was physically isolated in its own chamber. Differential pumping was also applied to the other quadrupoles. A total of four turbopumps were used to evacuate the mass spectrometer: q0 chamber, 250 L s-1 (Leybold, TW250S); Q1 chamber, 150 L s-1 (Leybold, Turbovac 151); q2 chamber, 700 L s-1 (Leybold, TW700); and Q3 chamber, 360 L s-1 (Leybold, Turbovac 361). The orifice (OR)/skimmer (SK) region was evacuated by a Leybold D8A mechanical pump. The 250 and 700 L s-1 turbopumps were augmented by a Leybold D16 mechanical pump. The 150 and 360 L s-1 turbopumps were backed by a second Leybold D8A mechanical pump. Standard triple-quadrupole electrical/electronic operation was applied with the exception of the 20-segment quadrupole/IMS cell (q2). Its rf voltage was supplied by an arbitrary waveform generator (Hewlett-Packard, 33120A) at a frequency of 1.26 MHz and amplified to 400-750 V peak-to-peak by an rf amplifier custombuilt by MDS Sciex. The axial drift field was decoupled from the radial rf field that enabled collisional focusing. The IMS cell comprised an enclosed 20-segment quadrupole with end caps serving as lens elements (Figure 1). The quadrupole segments were mounted on Delrin AF (copolymer of polyoxymethylene, formaldehyde homopolymer, acetal homopolymer, and poly(tetrafluoroethylene)) and thus were electrically insulated from each other. Insulating surfaces were positioned as far back as possible from the ion path to minimize surface charging. The conventional q2 offset (RO2) was set on the quadrupole lens, ST2, immediately upstream from q2. The axial drift potential (∆U), typically 20-160 V, was applied linearly across the 20 segments, U1 - U20, with a total length of 20.2 cm using a GW GPR-30H 10D dc power supply. Biasing of the optical components downstream from q2, IL3, SK2, and RO3, were referenced to U20 at constant differences in order to maintain a constant ion flight time (tf) between q2 and the detector. SK2 was biased by the ST3 channel on API 3000 electronics. A custom-built five-channel power supply was used to bias ion optic elements, IL2, U1, and IL3, that were not covered by conventional API 3000 electronics. A Keithley 610C electrometer was employed to monitor biasing of these additional ion optic elements. For ion mobility measurement, ions were injected into q2 by employing IQ2 as an ion gate. Ions were normally barred by biasing IQ2 to 70 V. To inject ions, IQ2 was lowered to -130 V for Analytical Chemistry, Vol. 77, No. 1, January 1, 2005

267

Figure 1. Schematic representation of the 20-segment quadrupole/IMS cell and the triple-quadrupole mass spectrometer: (a) general layout; (b) q2 region; and (c) details of the 20-segment quadrupole/IMS cell. q0, rf-only ion guide; Q1 and Q3, quadrupole mass analyzers; IQ1, IQ2, electrostatic lenses; IL2, IL3, additional enclosure lenses of drift cell; ST1, ST2, short rf-only prefilters; SK2, skimmer before Q3, replacing original ST3; q2, drift cell, dc offset set by U1; U1, U20, dc offsets of the first and last segments; V1, V2, pulse voltages set to -130 and 70 V, respectively; ∆U, E, axial field voltage and strength. 268

Analytical Chemistry, Vol. 77, No. 1, January 1, 2005

50 µs (Wavetek 50-MHz pulse/function generator amplified by a custom-built pulse power supply). Ion injection was typically repeated every 4-20 ms. Pulsing and rf biasing were monitored using a Hewlett-Packard 54600B 100-MHz oscilloscope. The output of the detector was fed to a Stanford Research System SR 430 multichannel scaler to permit recording of the arrival times (ta) of the ions. ta were measured at different ∆U with constant tf and at a time resolution of 10 µs. Typically, 300-3000 raw spectra were summed to improve signal/noise ratios. Ion mobility measurements were made with helium being the neutral gas at a typical pressure of 1 Torr. Helium was introduced directly into the 20segment quadrupole/IMS cell via a needle valve (Whitey); its flow rate was continuously monitored (Aalborg flow meter, GFM17). The helium pressure within q2 was measured by an MKS Baratron 722A with a stated accuracy of 0.5%. With a helium pressure of 1 Torr within q2, the monitored pressure in the chamber surrounding q2 was typically 1 mTorr (Leybold Vakuum GMBH, model ITR 100 16360). Operating pressures in the Q1 and Q3 chambers were 1-2 × 10-5 Torr. The skimmer (SK) immediately downstream from the orifice (OR) was permanently grounded. To ensure low initial ion energies, RO0 and ST2 were set at -1.0 and -2.0 V, respectively. The mobilities of peptide and protein ions were recorded at different OR values. Larger OR settings resulted in more extensive collionsional heating in the region and in general more extended conformations. Additional downstream collisions in q0 under zero applied axial field and high neutral number density conditions ensured complete loss of the internal energy gained in the lens region.20,21 Materials. Chemicals, peptides, and proteins were purchased from Sigma-Aldrich (St. Louis, MO). HPLC-grade water and dichloromethane were also from Sigma-Aldrich. Benzene and methanol were from Caledon Laboratories (Georgetown, ON, Canada). For mobility measurements, cesium nitrate solution was 100 µM in 1:1 water/methanol; bradykinin (BK) solutions were 5-50 µM in 1:1 water/methanol (plus 2% formic acid for maximizing the abundance of triply protonated BK); C60 solution was 10 µM in 99:1 dichloromethane/benzene; tryptic digest solution of cytochrome c was 0.6 mg of digest in 2 mL of 49:49:2 water/ methanol/acetic acid; and ubiquitin solutions were 10 µM in 1:1 water/methanol or pure water. Tryptic digestion of cytochrome c was performed by adding 10 µg of trypsin (Promega, sequencing grade) to 3.6 mg of cytochrome c in 1 mL of 50 mM NH4HCO3 in water and incubating at 37 °C for 24 h. The resulting digest was lyophilized and stored frozen until use. Ionization Methods. Cs+ and protonated proteins and peptides were generated from their respective solutions by means of ESI at a typical flow rate of 1 µL/min. C60•+ was generated by atmospheric pressure chemical ionization at a sample flow rate of 50 µL/min. The ion sources were standard components on the API 3000 triple-quadrupole mass spectrometer. Data Analysis. Ion mobilities (K) and collision cross sections (ΩT) were derived from the measured ta:

ta ) t d + t f

where td is drift time, the time that ions spent drifting in q2

(3)

td ) L/vd ) L/KE

(4)

L is the length of the segmented quadrupole (drift cell). As E ) ∆U/L, substituting into eq 3 gives

ta ) td + tf ) L2/K∆U + tf

(5)

For a set of measurements of ta under different ∆U and constant tf, plotting ta against 1/∆U gives a straight line whose slope is L2/K and intercept tf. To facilitate comparisons with K values in the literature, measured K values were converted to reduced mobility, Ko, using

KN ) KoNo where Ko and No refer to the values at standard temperature (273.15 K) and pressure (1 atm). From K, ΩT were calculated using eq 2. Theoretical fitting of ion mobility spectra was performed using a modified version of the software ATCAL provided by R. R. Hudgins and M. F. Jarrold. ATCAL calculates the diffusion coefficient, D, from K via the Einstein equation.1 Cross Section Predictions from Calculated Structures. The collision cross sections of selected ions were predicted from their calculated structures for comparison with cross sections obtained from mobility measurements. Three prediction models, projection,37 exact hard-sphere scattering (EHSS),38 and trajectory,39 were employed. Software for calculations based on these models was obtained under MOBCAL courtesy of M. F. Jarrold.40 RESULTS AND DISCUSSION Influences of the rf and Axial Fields. The rf field has a strong focusing effect and improves ion transmission through a quadrupole and the following aperture at a pressure g1 mTorr.20,41,42 Repeating selected mobility measurements in this study with the q2 rf field turned off resulted in no detectable ion signals. Comparisons with other studies are more difficult because not all reports gave the concentrations of the sample solutions and even some that did gave a wide concentration range. For proteins and peptides, a concentration of 10 µM gave ion mobility spectra of excellent signal/noise ratio (typically >50) on the 20-segment quadrupole/IMS instrument with 35-200 accumulated scans for the most abundant ions. Concentrations of 10 µM are among the lowest reported.14,25,29-32,43-47 Thus, it appears that the presence (37) von Helden, G.; Hsu, M. T.; Gotts, N. G.; Bowers, M. T. J. Phys. Chem. 1993, 97, 8182-8192. (38) Shvartsburg, A. A.; Jarrold, M. F. Chem. Phys. Lett. 1996, 261, 86-91. (39) Mesleh, M. F.; Hunter, J. M.; Shvartsburg, A. A.; Schatz, G. C.; Jarrold, M. F. J. Phys. Chem. 1996, 100, 16082-16086. (40) Jarrold, M. F. Website at http://nano.chem.indiana.edu/software.html. (41) Douglas, D. J.; French, J. B. J. Am. Soc. Mass Spectrom. 1992, 3, 398-408. (42) Lock, C. M.; Dyer, E. Rapid Commun. Mass Spectrom. 1999, 13, 432-448. (43) Valentine, S. J.; Counterman, A. E.; Clemmer, D. E. J. Am. Soc. Mass Spectrom. 1997, 8, 954-961. (44) Hoaglund, C. S.; Valentine, S. J.; Sporleder, C. R.; Reilly, J. P.; Clemmer, D. E. Anal. Chem. 1998, 70, 2236-2242. (45) Kinnear, B. S.; Hartings, M. R.; Jarrold, M. F. J. Am. Chem. Soc. 2001, 123, 5660-5667 (46) Sudha, R.; Kohtani, M.; Breaux, G. A.; Jarrold, M. F. J. Am. Chem. Soc. 2004, 126, 2777-2784.

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of the rf field does lead to improvement of ion transmission in the drift cell and result in good sensitivity. The magnitude of ∆U affects resolving power, defined as td/∆t (peak width at halfmaximum) ≈ ta/∆t; resolving power ranges from ∼7 for ∆U ) 20 V to ∼15 for ∆U ) 125 V (E ≈ 1-6 V cm-1). This is in line with the typical resolving power of 30 achievable in commercial atmospheric pressure ion mobility spectrometers that operate on E ≈ 300 V cm-1.14 The average radial energy of an ion, Er, within an rf-only quadrupole has been shown to be48,49

Er )

mω2ri2 2 m 2 v22(a ) 0,q) v21(a ) 0,q) + 8 2γ 2

(6)

⊥

where m is the ion mass, ω is the rf angular frequency, ri is the radial position of the ith ion, ν221 and ν222 are dimensionless parameters, q is the well-known dimensionless parameter in the Mathieu equation (q ) 2 eV/mω2ro2; V is the peak-to-peak rf potential, and ro is the center-to-rod distance; a ) 0 in an rf-only quadrupole), and γ⊥ ) 1/(2RT⊥)1/2 (T⊥ is the radial translational kinetic temperature, R ) k/m, and 1/γ⊥ is the most probable ion thermal speed). Collisions with helium result in collapse of the ion beam around the quadrupole axis.20,41,42 As a consequence, ri and therefore the potential energy (first) term in eq 6 are approximately zero

Er ≈ mν222(0,q)/2 γ⊥2

(7)

Figure 2. Reduced mobility of Cs+ in He at different fields: this work, (, 0.98 and ), 0.12 Torr He, 298 K, (3% uncertainty; 2, literature values from ref 51, 0.04-0.50 Torr He, 300 K, (3% uncertainty.

collisional heating available to the ions drifting in the 20-segment quadrupole/IMS cell. The collision between the ion and the neutrals has been viewed as a “slow” heating event.50 The frictional ion heating rate, Qi, is given by

Qi ) nµfvd2

(9)

where n is the ion number density and f is the collision frequency. As the helium number density is large, thermodynamic equilibrium is a good approximation. This means that the frictional ion heating rate is equal to the ion cooling rate, Q

Q ) 3kn[m/(m + mn)]f (Tn - T)

(10)

Substituting

Er ≈ kT⊥ν222(0,q)

(8)

where mn and Tn are the mass and the temperature of the neutral (helium). Equating eqs 9 and 10 gives

For q ) 0.4, ν222(0,q) ) 1.1800,49 T⊥ ) 298 K

3k(T - Tn) ) mnvd2

(11)

Er ≈ (1.381 × 10-23)(298)(1.1800) J ≈ 2.92 kJ/mol ≈ 0.0303 eV That is to say, the rf field’s contribution to the ion energy was negligible. ∆U was typically 20-160 V and L was 20.2 cm. Under a helium pressure of 1 Torr, this gives an E/N of (3.06-24.4) × 10-17 V cm2 or 3.06-24.4 Td. The choice of E/N is based on a compromise between the desire to operate under low-field conditions, typically estimated to be E/N e 6 Td for small ions, and to have sufficient sensitivity.1 Operating at 3-24 Td ensures a balance between accuracy and sensitivity. Plots of ta against 1/∆U are always linear (see later for an example) meaning that K is independent of E, a necessary outcome of low-field measurements. As collisions between ions and neutrals are the basis for ion activation for mass spectrometry, it is instructive to consider here the extent of (47) Liu, D.; Wyttenbach, T.; Carpenter, C. J.; Bowers, M. T. J. Am. Chem. Soc. 2004, 126, 3261-3270. (48) Baranov, V. I. J. Am. Soc. Mass Spectrom. 2003, 14, 818-824. (49) Baranov, V. J. Am. Soc. Mass Spectrom. 2004, 15, 48-54.

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Taking a typical vd of 40 ms-1 (C60•+ at an E of 1.2 V cm-1; see later), eq 11 gives

T - Tn ) 4.003 × 402/(3 × 1.381 × 10-23 × 6.022 × 1023 × 103) ) 0.257 K Thus, the axial field imposed for ion mobility measurements resulted in minimal collisional heating. In the following sections, mobility measurements of a number of relatively well-characterized ions are reported as hallmarks for the performance of the 20-segment quadrupole/IMS cell: Cs+. The mobility of this atomic ion has been measured over a wide E/N range and found to be independent ((3% deviation) of E/N in the range of 5-20 Td.51 Figure 2 shows a comparison of our experimental data with literature values. There is good agreement between the two sets of data. The combined data show (50) McLuckey, S. A.; Goeringer, D. E. J. Mass Spectrom. 1997, 32, 461-474. (51) Pope, W. M.; Ellis, H. W.; Eisele, F. L.; Thackston, M. G.; McDaniel, E. W.; Langley, R. A. J. Chem. Phys. 1978, 68, 4761-4762.

Table 1. Measured, Calculated, and Literature Cross Sections (Å2) of C60•+ and Protonated BK in Helium at 298 K ion C60•+

Figure 3. Ion mobility spectra of C60•+ (2.00 V cm-1 in 1.05 Torr He, 298 K). The theoretical distribution is based on a consideration of diffusion that predicts peak shape.

measured

calculated

123 ( 1

119a

[BK + H]+

248 ( 6

[BK + 2H]2+

242 ( 4

[BK + 3H]3+

275 ( 5 293 ( 5

121b 125c 241a 274b 264c 244a 278b 266c 281a 316b 312c

literature 126d 245,e 239f 255,g 240f 284f

a Projection modeling.37 b EHSS modeling.38 c Trajectory modeling.39 d Reference 60. e Reference 4. f Reference 58. g Reference 56.

cross sections using a C60•+ structure predicted by Petrie.52 The measured precision for the cross section of C60•+ from three replicates was (1 Å2. However, the average precision of IMS measurements in our laboratory is (2%. This means that both literature cross sections and all three calculated cross sections are within or just on the edge of experimental uncertainties. Singly, Doubly, and Triply Protonated Bradykinin. BK is an important nonapeptide (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) that has frequently been used as a model peptide in gas-phase ion chemistry and mass spectrometry studies.4,53-57 Several ion mobility studies have concluded that protonated BK can exist as multimers or have multiple conformations.44,58,59 Figure 5a shows measured ion mobility spectra of [BK + H]+, [BK + 2H]2+, and [BK + 3H]3+ at a low OR of 30 V at an E of 1.95 V cm-1. Comparison with theoretical distributions (shown in dotted lines) reveals that [BK + H]+ comprises a minimum of three structures, [BK + 2H]2+ one structure, and [BK + 3H]3+ two structures. As far as we know, this is the first report of more than one structure observed for [BK + 3H]3+. By contrast, ion mobility spectra measured under high OR values are simpler and favor the structures having the longest arrival times. Figure 5b shows ion mobility spectra of [BK + 3H]3+ recorded under OR ) 5-40 V. Mass spectral windows in the range of 1,059-1,065 Th for [BK + H]+ at OR ) 5-100 V are shown in Figure 6a. The existence Figure 4. (a) Ion mobility spectra of C60•+ at different fields (1.05 Torr He, 298 K); (b) arrival time of C60•+ versus 1/∆U (1.05 Torr He, 298 K, measurement uncertainty: (2%).

a constant Ko value of 18 cm2 V-1 s-1 for Cs+ in helium within the E/N range of ∼1-20 Td. C60•+. This is another well-characterized ion.39 Figure 3 shows a mobility spectrum of C60•+ at an E of 2.00 V cm-1, pressure of 1.05 Torr, and temperature of 298 K. The dotted trace shows the theoretical distribution of C60•+ based on diffusion.1 Figure 4a shows a plot of various mobility spectra measured in the E range of 0.90-6.25 V cm-1 at a pressure of 1.05 Torr and temperature of 298 K; Figure 4b is a plot of ta versus 1/∆U, demonstrating the linear relationship of eq 5. From the slope, K and then ΩT were calculated (the latter via eq 2). Table 1 shows a comparison of the measured cross section with literature data and calculated

(52) Simon, P. using B-LYP/TZP from Amsterdam Density Functional 2000 program package; TZP basis set was formerly known as “Type IV” basis set. (53) von Helden, G.; Wyttenbach, T.; Bowers, M. T. Science 1995, 267, 14831485. (54) Schnier, P. D.; Price, W. D.; Jockusch, R. A.; Williams, E. R. J. Am. Chem. Soc. 1996, 118, 7178-7189. (55) Price, W. D.; Schnier, P. D.; Williams, E. R. Anal. Chem. 1996, 68, 859866. (56) Gill, A. C.; Jennings, K. R.; Wyttenbach, T.; Bowers, M. T. Int. J. Mass Spectrom. 2000, 195/196, 685-697. (57) Ewing, N. P.; Pallante, G. A.; Zhang, X.; Cassady, C. J. J. Mass Spectrom. 2001, 36, 875-881. (58) Counterman, A. E.; Valentine, S. J.; Srebalus, C. A.; Henderson, S. C.; Hoaglund, C. S.; Clemmer, D. E. J. Am. Soc. Mass Spectrom. 1998, 9, 743759. (59) Wyttenbach, T.; Kemper, P. R.; Bowers, M. T. Int. J. Mass Spectrom. 2001, 212, 13-23. (60) von Heldon, G.; Wyttenbach, T.; Bowers, M. T. Int. J. Mass Spectrom. 1995, 146/147, 349-364.

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Figure 6. (a) Mass spectra of bradykinin ions at different orifice voltages (50 µM in water); (b) ion mobility spectra of bradykinin ions at different m/z (3.00 V cm-1 in 1.09 Torr He, 298 K).

Figure 5. (a) Experimental and theoretical ion mobility spectra of protonated bradykinin ions (1.95 V cm-1 in 1.04 Torr He, 298 K, OR ) 30 V); (b) ion mobility spectra of [BK + 3H]3+ at different orifice voltages (3.00 V cm-1 in 1.09 Torr He, 298 K).

of a peak at 1061.2 Th (and less prominently a second one at ∼1062.2 Th) is evident at OR e 40 V. At OR ) 100 V, the peaks at 1060.8, 1061.8, and 1062.8 Th are principally [BK + H]+ that contains zero, one, and two 13C atoms, respectively. At OR e 40 V, these peaks contain a component of [BKn + nH]n+ (n ) 2 or higher), which are efficiently dissociated into [BK + H]+ under larger OR values (more energetic collisions in the lens region). The increasing presence of [BK2 + 2H]2+ with decreasing OR is evidenced by the increasing abundance of the 1061.2 Th ion, 13C[BK2 + 2H]2+ ([BK2 + 2H]2+ that contains one 13C atom). Despite the employment of resolution considerably better than unit mass, a significant fraction of ions under the 1061.2 Th envelope is [BK + H]+. Interpretation of the presence of multimers in the mobility spectrum of [BK + H]+ is strengthened by the mobility spectra of 1060.6, 1060.9, and 1061.1 Th shown in Figure 6b at OR values of 30 and 60 V. The resolving quadrupole was operating on a resolution comparable to the one shown in the mass spectra. 1060.6 Th is on the left shoulder of the [BK + H]+ peak, 1060.9 Th the right shoulder, and 1061.1 Th the apex of the 13C-[BK2 + 2H]2+ peak. It is apparent that the ratio of the area under the two peaks of shorter arrival times to the area under the peak of the 272 Analytical Chemistry, Vol. 77, No. 1, January 1, 2005

longest arrival time increases with increasing m/z among the three values. This phenomenon is in accordance with the expectation that the proportion of [BKn + nH]n+ in the mass-selected ions would increase with increasing m/z among the three values being examined. For [BK + 3H]3+, attempts to determine whether the peak at the shorter arrival time is a multimer or not were inconclusive because resolving the all-12C ion from the 13C1 ion requires much higher resolution than it is possible for our resolving quadrupole. The measured, calculated, and literature cross sections for [BK + H]+, [BK + 2H]2+m and [BK + 3H]3+ are also given in Table 1. The measured cross section shown for [BK + H]+ is that for the monomer, which gives the peak of the longest drift time. Two cross sections were given for [BK + 3H]3+. The one with the smaller value is only valid if the peak that has the shorter drift time is due to monomer that has a more compact conformation relative to that conformation giving the peak at the longer drift time. There is good to acceptable agreement between the measured cross sections and literature cross sections. The calculated values were population-averaged cross sections based on lowest energy structures predicted by HF/6-31G calculations.61 For BK, EHSS modeling produces the largest calculated cross sections, followed by trajectory and projection modeling. Our (61) Guo, Y.; Orlova, G.; Rodriquez, C. F.; Wang, J.; Javahery, R.; Ling, Y.; Thomson B. A.; Hopkinson, A. C.; Siu, K. W. M. Proceedings of the 51th American Society for Mass Spectrometry Conference, Montreal, PQ, June 7-12, 2003.

Table 2. Measured and Literature Cross Sections (Å2) of Tryptic Peptides from Cytochrome c in Helium at 298 K singly protonated

a

doubly protonated

peptide (Mr)

expt

lit.a

AK (217) KK (274) HK (283) LR (287) GKK (331) YTK (410) AGIK (387) ANTE (433) KANTE (562) Ac-GDVEK (589) GITWK (605) IFVQK (634) TGPNLH (638) YIPGTK (678) EDLIAY (723) MIFAGIK (779) EDLIAYLK (964) TGPNLHGLFGR (1168) TEREDLIAYLK (1350) TGQAPGFTYTDANK (1470) EETLMEYLENPK (1495) KTGQAPGFTYTDANK (1598) EETLMEYLENPKK (1623)

85 ( 1 97 ( 1 96 ( 1 109 ( 1 111 ( 1 127 ( 2 128 ( 2 129 ( 3 155 ( 2 165 ( 2 170 ( 2 184 ( 3 165 ( 2 186 ( 3 193 ( 2 212 ( 3 243 ( 2 267 ( 3 309 ( 4 300 ( 3 316 ( 7 322 ( 3 330 ( 4

87.01 97.4 ( 0.6 99.0 ( 0.8 110.0 ( 1.4 111.7 ( 1.2 127.0 ( 1.1 130.2 ( 0.4 128.3 154.0 ( 0.3 163.2 ( 1.3 169.3 ( 0.6 182.0 ( 0.5 164.3 ( 0.3 183.8 191.7 ( 0.3 207.1 ( 0.4 257.7 286.7 309.0

expt

161 ( 2 176 ( 2 180 ( 1 184 ( 2 202 ( 4 204 ( 2 237 ( 1 268 ( 3 307 ( 1 297 ( 3 312 ( 3 316 ( 2 327 ( 3

lit.a

172.8 189.0 ( 3.6 207.0 ( 1.3 267.6 ( 0.7 297.6 ( 2.2 312.7 ( 0.8

Reference 25.

Figure 7. Comparison of measured cross sections of singly and doubly protonated tryptic peptides between this work and literature values from refs 25 and 62.

measured cross sections appear to be closer to theoretical cross sections calculated using trajectory and projection modeling. Tryptic Peptides. Clemmer and co-workers measured and tabulated the collision cross sections of a large number of tryptic peptides from common proteins.25,62 We measured the cross sections of 23 singly and doubly protonated tryptic peptides of cytochrome c and compared them with those reported by the Clemmer group.25 The results are shown in Table 2 and Figure 7. As in previous examples, there is good agreement between the cross sections measured here in this study and literature cross sections. Peptides that have fewer than five residues are typically only singly protonated. Longer peptides have increased propensity for double protonation, probably a consequence of better charge solvation with increasing number of residues. Interestingly the (62) Clemmer, D. E. Web site at http://www.indiana.edu/∼clemmer/.

cross sections of singly and doubly protonated peptides are comparable for most tryptic peptides that are longer than six residues. There appear to be larger relative differences in cross sections between singly and doubly protonated peptides when they are shorter. Ubiquitin (UB). This is a small protein with 76 residues and no disulfide bonds. The absence of disulfide bonding means that the protein conformation is relatively flexible. A number of UB charge states have been reported to exist as multiple conformers.43,59 For this reason, we picked UB as a potentially useful model system to test the performance of the 20-segment quadrupole/ IMS instrument. Figure 8a shows a comparison of the ion mobility spectra of [UB + 6H]6+ and [UB + 7H]7+ under OR ) 30-200 V. At OR ) 30 V, the former appears as three and the latter two peaks. The peaks at shorter drift times vanish at larger OR values, in accordance with interpretations of more compact conformations opening up to more open structures with increasing injection energies.43,59 Figure 8b displays ion mobility spectra of the various UB charge states obtained under OR ) 30 V after converting the time axis to cross section. Two UB dimer ions, [UB2 + 9H]9+ and [UB2 + 11H]11+, are apparent in the mass spectrum of UB shown in Figure 8c; their mobility spectra and that of [UB2 + 13H]13+ in terms of cross section per UB unit are also included in Figure 8b. As expected, increasing charge increases the extent of Coulomb repulsion, which leads to an increasingly open conformation. Comparing the experimental and theoretical distributions, it appears that [UB + 9H]9+ to [UB + 11H]11+ contain only one principal conformer per charge state. The number of conformers appears to maximize at [UB + 5H]5+ or [UB + 6H]6+. The experimental distribution for [UB + 5H]5+ shows one broad peak that contains several unresolved conformers. By contrast, [UB + 6H]6+ appears to comprise three resolved groups of Analytical Chemistry, Vol. 77, No. 1, January 1, 2005

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Figure 8. (a) Ion mobility spectra of [UB + 6H]6+ and [UB + 7H]7+ at different orifice voltages (4.00 V cm-1 in 1.02 Torr He, 298 K); (b) ion mobility spectra of protonated UB ions at low orifice voltage (4.00 V cm-1 in 1.02-1.06 Torr He, 298 K, OR ) 30 V); V representing literature values from ref 62; theoretical distributions showing diffusion-limited peak shapes from single conformers; and (c) mass spectrum of UB (10 µM in water, OR ) 30 V).

conformers; within each group there appears several unresolved conformers. [UB2 + 9H]9+ has a cross section per UB unit that is comparable to that of [UB + 4H]4+. Interestingly, [UB2 + 11H]11+ has a cross section per UB unit that is comparable to the cross section of the [UB + 6H]6+ group of conformer with the intermediate size. Further increase in charge appears to narrow the distribution and decrease the number of conformers. [UB + 7H]7+ has only one major group of conformers and a second minor group. [UB + 8H]8+ appears to comprise a group of unresolved conformers. As noted before, increasing the charge state beyond [UB + 8H]8+ appears to decrease the number of conformers to ∼1, probably a consequence of the increasingly open UB geometry due to Coulomb repulsion. 274 Analytical Chemistry, Vol. 77, No. 1, January 1, 2005

CONCLUSION The 20-segment quadrupole/ion mobility spectrometer appears to have attributes of good sensitivity, high resolution, and accurate cross sections as exhibited by experiments with Cs+, C60•+, BK ions, protonated tryptic peptides, and UB ions. The combination of high pressure (1 Torr) and low axial field (20-160 V per 20.2 cm) results in negligible internal excitation of the ions as a result of the applied rf and axial fields.

ACKNOWLEDGMENT We thank Drs. Yun Ling, Robert R. Hudgins, Simon Petrie, Christopher F. Rodriquez, Galina Orlova, Alan C. Hopkinson, and

Vladimir I. Baranov for helpful discussion and advice. The expertise of York University’s machine shop and MDS Sciex’s electronic shop is gratefully acknowledged. This research was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada, MDS Sciex, and York University. Parts of this work were presented at the 51st ASMS Conference,

Montreal, PQ, June 7-12, 2003, and the 52nd ASMS Conference, Nashville, TN, May 23-27, 2004. Received for review July 13, 2004. Accepted October 8, 2004. AC048974N

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