Detection of High-Mass Biomolecules in Fourier Transform Ion

Apr 1, 1994 - Atmospheric Pressure MALDI-Fourier Transform Mass Spectrometry. Katherine ... Dey , Salvador J. Pastor , and Charles L. Wilkins. Analyti...
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Anal. Chem. 1994,66, 1583-1687

Detection of High-Mass Biomolecules in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: Theoretical and Experimental Investigations Touradj Solouki, Kent J. Gllllg, and David H. Ruesell’ Department of Chemistry, Texas A&M University, College Station, Texas 77843 A simple modification to an ion cyclotron resonance (ICR) cell for detectionof high-massions with large spatial and kinetic energy distributions is described. The modification consists of a copper wire positioned inside the ICR cell rUaning parallel to the magnetic field lines. The wire acts as an ion guide to position the ions at the exact center of the ion cell. Ion trajectories are calculated using SIMION,and the predictions based on the calculationsare compared with the experimental results. The results presented in this paper show that the ionguide ICR cell can be used to detect singly charged biomolecule ions of up to 157 OOO Da. The technique of matrix-assisted laser desorption ionization (MALDI) has greatly expanded the scope of biological mass spectrometry.IJ For example, MALDI has been used to produce intact ions of peptides and proteins with molecular masses in excess of 200 000 Da,3 and the analysis can be performed at the low picomole to femtomole level^.^ There has been considerable interest in combining MALDI with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. The MALDI/FTICR experiment would combine the high-resolution and high-precision mass measurement capabilities of FTICR with the ability to ionize very large, polar biomolecules. To date, however, these efforts have been complicated by the high kinetic energiesof the laser-desorbed ions.596 For example, gramicidin S and bovine insulin [M HI+ ions are desorbed with approximately 3 and 11 eV of axial kinetic energies, respecti~ely.~ The large kinetic energy of laser-desorbed ions complicates both ion trapping and ion detection.6 Numerous experimental approaches have been taken to improve high mass ion trapping efficiency and mass resolution in ICR instr~ments.~-l~ We have demonstrated that high

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(1) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. In?. J. Mass Spectrom. Ion Processes 1987, 78, 53. (2) Karas, M.;Hillenkamp, F. Anal. Chem. 1988,60, 2288. (3) Chan, T. W.; Colburn, A. W.; Derrick, P. J. Org. Mass Spectrom. 1992.27, 53. (4) Strobel, F. H.; Solouki, T.; White, M. A.; Russell, D. H. J. Am. Soc. Mass Spectrom. 1991, 2, 91. (5) Bcavis. R. C.; Chait, B. D. Chem. Phys. Lett. 1991, 181, 402. (6) Hanson, C. D.;Kercly. E. L.;Castro, M. E.; Russell, D. H. Anal. Chem. 1989, 61, 2040. (7) Solouki, T.; Russell, D. H. Proc. Natl. Acad. Sci. U S A . 1992, 89, 5701. (8) May, M.A,; Grosshans, P. 9.; Marshall, A. G. In?. J. Mass Spectrom. Ion Processes 1992, 120, 193. (9) Castoro, J. A.; Wilkins, C. L. AMI. Chem. 1993, 65, 2621. (10) Rempel, D. L.;Grew R. P.;Gross, M.L. Inr. J. MassSpecrrom. Ion Processes 1990,100, 381. ( 1 1 ) KCter,C.;Castoro, J.A.; Wi1kins.C. L.J. Am. Chem.Soc. 1992,114,7572. (12) Rempel, D. L.; Gross, M. L. 1.Am. Soc. Mass Spectrom. 1992,3, 590. (1 3) Guan, S.;Wahl, M.C.; Wood, T. D.; Marshall. A. G. Anal. Chcm. 1993,155, 1753.

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trapping potentials and/or collisions greatly enhance the performance of FTICR.’ For instance, the absolute abundance of high m/z ions trapped inside the ICR cell increases as the trapping voltage is increased (See Figure 5 of ref 7). Although the theoretical upper mass limit for thermal energy ions in a 7-T magnet FTICR is -300 000 Da,* the highest reported singly charged ion trapped in an ICR cell is the dimer ion of carbonic anhydrase (m/z 58 000) with a signal-to-noise ratio of less than Rempel et al. used a “scaling technique” to demonstrate that increasingthe number of ions trapped in the cell increases both theS/N ratio and the resolution.10 Wilkins and co-workers used a gated trap method and sugars as “comatrix” compounds to obtain high-resolution mass spectra of bovine insulin (at MW = 5733.6), ubiquitin (MW = 8564.9) and cytochrome c (MW = 12 360.1).11 Mass resolutions of 11 580 and 12 000 were reported for bovine insulin at m/z 5733.69 and cytochrome c at m/z 12 349.0, respectively. An important consideration for the performance of ICR is the initial position of the ions in the cell and the initial ion kinetic energy distribution. The latter consideration is of particular importance because recent reports suggest that the kinetic energies of MALDI ions increase as the mass of the ions increases.’ Rempel and Gross have recently developed a quistor mode (combined high electric field and ion-neutral collisions) FTICR experiment to deal with high kinetic energy ions that may have direct applications to high-mass measurements.12 Marshall and co-workers developed an ion axialization method that utilizes ion-neutral collisions for ion cloud compression and ion c o ~ l i n g . ~Each ~ J ~ of these approaches has the objective of positioning the ions in the center of the cell to optimize ion trapping and detection. Recently, Hendrickson and Laude used concepts first introduced by Baker and Hasted” to perform a detailed (SIMION) analysis of the potential well created by an electron beam in an ICR cell.I6 Their results illustrate the point that electron impact ionization is ideal for achieving high performance of the ICR due to two factors: (i) ions are formed only alongthe center line of the ion cell, and (ii) during the beam“on” cycle, the ions are trapped in a deep electrostatic well. We decided to examine the effects of such an electrostatic well on the trapping of ions formed by MALDI in an ionguide cell. This cell is equipped with a wire ion guide that (14) Guan, S.; Xiang, X.; Marshall, A. 0. In?. J. Mass Spectrom. Ion Processes 1993, 124, 53. (IS) Baker, F. A.; Hasted,J. B. Philos. Trans. R. Soc. 1966, 261, 33. (16) Hendrickson, C. L.; Hadjarab, F.; Laudc, D. A., Jr. Proceedings of the 41th ASMS Conferenceon Mass Spectrometry and Allied Topics;San Francisco, CA, 1993; p 173a.

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focuses high kinetic energy/high mass biomolecule ions into the center of the cell. The wire ion guide is positioned parallel to the magnetic flux lines. Marshall and co-workers have also used an ion guide to transport ions from an external ion source into the ICR cell;" our design differs in that the wire ion guide is suspended inside the ICR cell as opposed to being external to the cell. To investigate the ion motions, the SIMION program was used to calculate ion trajectories and equipotential lines created inside the cell by the wire.I8 The MALDI/FTICR mass spectra of bovine serum albumin (M W = 66 430) and transferrin (MW = 78 685) obtained by using the ion-guide cell are reported. The results reported demonstrate that the ion-guide ICR cell facilitates ion trapping, e.g., high-mass ions as large as 157 000 Da formed by MALDI are trapped and detected.

EXPER I MENTAL SECT1ON Instrumentation. The experiments described herein were performed on a 7-T Fourier transform ion cyclotron resonance mass spectrometer. A detailed description of the instrument has been given previo~sly.~J~ Briefly, the spectrometer consists of an Extrel FTMS-2001 data system, a 7.0-T, 15-cm, roomtemperature bore superconducting Oxford magnet, and a home-built cylindrical ion cell. The vacuum chamber is pumped by two cryopumps (Model RPK 900 LeyboldHeraeus) with a maximum pumping speed of 2600 L/s and a minimum pumping speed of 300 L/s for H20 and He, respectively. The insertion lock used for sample introduction is differentially pumped by a 285 L/s oil diffusion pump. The typical ion cell vacuum during MALDI is in the mid 10-8 Torr range as measured with a Bayard-Alpert ion gauge. Desorption/ionization is performed using a Laser Science, Inc. cartridge type pulsed N2 laser (Model VSL-33ND) operated at a wavelength of 337.1 nm with a pulse width of 3 ns. The SIMION program PC/PS2 Version 4.0 was used to calculate the ion trajectories and equipotential lines inside the cell. Cell Design and Ion Detection. A 0.003-in.-diameter copper wire is suspended between the ion cell trapping plates. The wire runs parallel to the magnetic field lines and is connected to a dc voltage output of the ion cell controller. The standard experimental pulse sequence used for FTICR was revised to place a variable static voltage on the ion-guide wire at different times in the event sequence. To trap the positive ions, the front trapping plate is biased at 0.0 V when the laser is fired and this potential remains at 0.0 V for 50-300 ps. The rear trapping plate (trapping plate away from the sample insertion probe) is maintained at +9 V, and the wire ion guide is biased at a negative potential. Typical conditions required to guide the high-mass positive ions into the cell are -2 to -1 V applied to the wire ion guide. At -50-300 ps after the ions are transferred into the cell, both front and rear trapping plates are raised to +9 V. Because the ion's motion is strongly influenced by the potential applied to the ion-guide wire a positive potential is applied to the wire ion guide during the (17) Limbach, A. L.;Marshall, A. G.; Wang, M. In?. J . Mass Specrrom. Ion Processes 1993, 125, 135. (18) Dahl, D. A.; Delmore, J. E. SIMION PC/PS2 V 4.0, Idaho National Laboratory, Idaho Falls, ID, 1988. (19) Solouki, T.; Russell, D. H. Appl. Spectrosc. 1993, 47, 211.

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radio frequency excite-to-detect event. Typically a potential of 1-5 V is sufficient to force the ions outward. Ions are excited by a chirp excitation with a radio frequency field of 200,V from 0 to 200 kJ3z and a sweep rate of 200 Hzlps. Ion detection is performed without signal averaging with 16K time-domain data points. To account for the frequency shifts associated with the electric field changes, the mass spectra are calibrated using high mass ion calibrants. SamplePreparation. Bovine serum albumin and transferrin were dissolvedin ethanol (0.5 mg/mL), and 1% triflouroacetic acid was added. A binary matrix composed of 4-nitroaniline and fructose was used for MALDI. The ethanol solutions of 10 mg/mL 4-nitroaniline and 10 mg/mL fructose were prepared, and a 0.2-pL aliquot of each solution was deposited on the sample probe and air-dried for mass analysis. The amount of analyte on the probe tip was approximately 1-2 fmols.

RESULTS This paper describes the results of studies designed to evaluate a wire ion-guide ICR cell. The equipotential lines created inside the cell by the guide wire and trajectories of ions formed by MALDI are calculated using the SIMION program. A comparison between the experimental results and theoretical predictions is presented. Parts a and b of Figure 1 contain plots of the equipotential lines for a 9-V static potential placed on the trapping plates of a cylindrical ICR cell without and with the wire ion guide. In Figure l a the trapping field at the midpoint of the cell is -0.5 V. The wire ion guide in Figure l b is at ground, and a potential of 9 V is applied to the trapping plates. The potential at the midpoint of the wire ion-guide cell and more than 50% of the region inside the cell (Figure lb) is at a potential of less than 0.1 V. Parts a and b of Figure 2 contain trajectories for ions of m / z 200 000 with total translational energies, Le., Et ( x , y, z ) , of 5 eV in the cylindrical cell and in the cylindrical cell with an ion guide. The initial position of both ions are 1 cm inside the cell moving at 45O and 50' angles with respect to the magnetic field lines (z in ICR conventions). Note that both ions depicted in Figure 2a are lost to the ICR cell plates. However, in Figure 2b using the wire ion-guide cell and the same initial position, direction, and kinetic energies, both ions are trapped. Figure 3 shows ion trajectories in the wire ion-guide cell with the ion guide set at a -70 V with other parameters the same as for Figure 2a. The ion motion is determined predominately by the potential applied to the ion guide. For example, the cyclotron radius of the ion is smaller when the wire ion guide is at -70 V. It is interesting to note that in Figure 3 the ion motion in the z direction is also restricted. Thus, positive ion trapping is more efficient with higher negative voltages on the wire ion guide. Parts a and b of Figure 4 contain single laser shot MALDI/ FTICR mass spectra of bovine serum albumin (MW = 66 430) and transferrin (MW = 78 685) obtained using a 4-nitroaniline/fructose binary matrix in the ion-guide cell. For the data shown in Figure 4a,b only ions with masses higher than 550 amu were excited. In Figure 4a, the observed signal corresponds to bovine serum albumin. In Figure 4b signals

Figure 2. Ion trajectories calculated with SIMION for ions with mlz 200 000 and total translationalenergies of 5 eV in a 7-T magnet shown for (a, top) a cylindrical cell and (b, bottom) a cylindrlcai cell equipped with a wire lon guide. The trapping plates are biased at +9 V, and the initial positions of both ions are 1 cm inside the cell moving at 45' and 50' angles with respect to the magnetic field lines.

Figure 1. SIMION plots of trapping fields in a cylindrical ICR cell (a, top) without and (b, bottom) with wire ion guide. Trapping plates are biased at 4-9 V and the wire ion guide in (b) is at ground potential.

from monomer and dimer ions of transferrin are observed. These ions cannot be observed without the ion guide.

DISCUSSION The results presented here show that singly charged ions of up to 157 000 Da can be detected by FTICR, but steps must be taken to ensure that the ions are initially positioned at the center of the cell. Ions formed by MALDI have substantial axial and radial kinetic energies, and the large axial kineticenergyof the ions makes trapping in thez direction of the ICR cell difficult. Similarly, the high radial kinetic energy causes random ion dispersion in the x-y plane throughout the cell, and it is difficult to "drive" these randomly dispersed high kinetic energy ions into a coherent package for detection by ICR.6 ~

(20) Hanson, C. D.; Castro, M. E.; Kerely, E. L.;Russell, D. H. Anal. Chem. 1990, 62. 520.

Ion trajectories obtained by SIMION show that ion motion is restricted by the ion guide in both the x-y plane and the z direction; consequently, high-mass ions are trapped by the ion-guide wire cell. Furthermore, the trajectory calculations show that the ions orbit around the ion-guide wire in the center of the cell; consequently, detection of the high-mass ions that are redirected and positioned in the center of the cell is possible. The wire ion guide has the same effect as the "field-corrected" and/or "screened" cell in changing the trapping electric field (E)throughout the cell.20.21 The experimental data are in excellent agreement with the theoretical findings. The high-mass detection limit of FTICR is extended from -20 000 for a cylindrical cell to 157 000 for an ion-guide cylindrical cell. However, an important experimental requirement for the excitation and detection of high-mass ions is that the ions must be forced away from the ion guide before excitation by the radio frequency pulse. A positive voltage applied to the wire ion guide during the

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(21) Wang, M.; Marshall, A. G. Anal. Chem. 1990,62, 515. (22) Rempel. D. L.;Gross, M. L. Anal. Chem. 1984, 56, 2748. (23) (a) Solouki, T.; Russell, D. H. Pittsburgh Conference and Exposition on Analytical Chemistry and Applied Spectroscopy; Atlanta, GA, 1993;p 447. (b) Solouki,T.; Gillig, K. J.; Russell, D. H. Rapid Commun. MossSpectrom. 1994, 8, 26. (24) Covey, T.; Douglas, D.J. J. Am. Sac. Mass Spectrom. 1993, 4, 616. (25) Henry, K. D.;Quinn, J. P.; McLafferty, F. W. J. Am. Chem.Soc. 1991,113, 5447. (26) Henry, K. D.; Williams, E. R.; Wang, B. H.; McLafferty, F. W.; Shabanowitz J.; Hunt, D. F. Proc. Narl. Acad. Sci. U.S.A. 1992, 86, 9075. (27) Loo,J. A.; Quinn, J. P.; Ryu, S. I.; Henry, K. D.: Senko, M. W.; McLafferty, F. W. Proc. Null. Acad. Sci. U.S.A. 1992, 89, 286.

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the applied electric potential on the ion guide must be reversed (positive voltage for the positive ions); this is because the E field exerted on the trapped ions is dominated by the ionguide wire potential. Because the electric field is changing inside the ICR cell, careful calibration of mass spectra is required. The mass calibration equation for the FTICR contains terms for the B and E fields?*

Flgure 9. SIMION plot of an ion trajectory with mlz 200 000 in a 7-1 magnet and cyiindflcal ICR cell equlpped with an ion guide. A -70 V of static potenthi Is placedon the wire Ion gukle, and the other variable parameters are the same as for Figure 2a.

Flgwe 4. The single laser shot MALDI/FTICR mass spectra of (a, top) bovlne serum albumin (MW = 66 430) and (b, bottom) transferrin (MW = 78 685). A mixture of 4-nitroanlilne and fructose is used as the binary matrlx.

excitation event is needed to drive the positively charged trapped ions toward the excitation plates. This requirement suggests that the ions are initially under the influence of the E field of the ion guide rather than the radio frequency excitation electric field. Even though the successful trapping of high-mass ions requires the use of high potentials of opposite polarity on the copper wire, in the excite and receive events the polarity of 1588 Analytical Chemlsity, Vol. 60, No. 9, M y 1, 1994

kl and kz are constants. The magnetic field produced by the superconducting magnet remains stable over a long period of time, but the second term has a strong effect on the calibration, especially at high mass (low frequency);i.e., as the frequency decreases (or mass increases) the l/fz term becomes more important. The consequence is that accurate high-mass measurement requires calibration tables obtained from highmass calibrants. Moreover, shot-to-shot frequency shifts of up to f30 Hz that are frequently observed in FTICR/MALDI become more important in accurate molecular weight determination of high-mass ions. Thus, performance of FTICR mass spectrometry for mass measurement of high-mass ions can be enhanced by the use of internal calibrants and by acquiring a spectrum from a single laser Although detection of high-mass ions in an ICR cell is inspiring, there are limitations to the experiment that future work must address. For example, we can trap ions such as gramicidin S ( m / z = 1142) for up to several hundred seconds in the ion-guide cell but large ions such as bovine serum albumin can only be trapped for a few hundred microseconds. The larger collision cross section of bulky, high-mass ionsZ4 may contribute to the mechanism of high-mass ion loss to the wire ion guide. The equipotential lines observed in Figure 1 illustrate that the wire ion guide alters the electric field experienced by an ion. Because the voltage applied to the wire can be varied during an experiment, ions can be redirected. For example, high-mass, high kinetic energy positive ions are better focused by application of a higher negative voltage on the wire with respect to the trapping plates (Figure 3). Furthermore, the inhomogeneity of the trapping electric field is reduced by the wire ion guide. The effect of the inhomogeneous trapping electric field is that two ions of the same m / z value that have equal axial translational energies but different radial velocities will experience different electric fields and will have two distinctly different ion motions resulting in slightly different measured cyclotron frequencies. Thus, the wire ion guide serves a dual purpose as an ion-focusing device and as a device to extend the region with a homogeneous dc electric field. CONCLUSIONS Ion trajectories calculated by SIMION and experimental results presented in this paper demonstrate that the use of an ion-guide cell is advantageous for trapping and detection of high-mass (up to 150 kDa), high kinetic energy ions formed by MALDI. The mass resolutions at these very high mass ranges are far below the resolving power obtainable by electrospray ionization methods. For example, McLafferty and co-workers demonstrated a mass resolution of 50 00080 000 for ions as large as 17 kDa formed by electrospray

ionization (ESI) coupled with FTICR.25 ESI has the unique advantage of generating molecular ions that are multiply charged and can be detected in the more convenient massto-charge ( m / z ) range of 500-2000.2628 Nonetheless, the excellent sensitivity and mass range offered by MALDI/ FI'ICR exceeds that of ESI/FTICR, offsetting the lower resolution. The experimental results suggest that the difficulties associated with MALDI/FTICR analysis of highmass ions are primarily due to the high kinetic energy of desorbed ions formed by MALDI. These results are consistent with the measured kinetic energy of ions formed by MALDI5329930 and previous reports on high-mass FTICR.7*9J1.31In conclusion,we have demonstrated that using a wire ion guide high-mass, high kinetic energy ions produced by MALDI are trapped. Although the wire ion guide improves the ion trapping and ion detection by FI'ICR, issues related to accurate mass calibrations at high mass are not completely resolved. The (28) Laude, D.A. Anal. Chem. 1992,64, 569. (29) (a) Ens, W.; Mao, Y.; Maycr, F.; Standing, K. G. Rapid Commun. Mass Spectrom. 1991,5177. (b) Zhou, J.; Ens,W.; Standing, K.G.;Verentchikov, A. Rapid Commun. Mass Spectrom. 1992, 6, 71. (30) (a) Kinsel, G. R.; Gillig, K. J.; Russell, D. H., manuscript in preparation. (b) Spengler, B.; Bdkelmann, V. Nucl. Instrum. Methods Phys. Res., in press. (31) Hanson, C. D.; Castro, M. E.;Russell, D. H.; Hunt, D. F.; Shabanowitz, J. In Fourier Transform Mass Spectrometry: Evaluation, Innovation, and Applicarlon; Buchanan, M. V., Ed.;ACS Symposium Series 359; American Chemical Society: Washington DC, 1991; pp 100-1 15.

wire ion guide changes the electric field experienced by an ion inside the cell, and the resulting frequency shifts must be accounted for by calibration of the mass spectra. Because the observed cyclotronfrequencyof a high-massion is signikantly lower than cyclotron frequency of a low-mass ion, similar frequency shifts at high mass correspond to larger shifts in the mass assignment. Thus, shot-to-shot variations in the observed cyclotron frequency of laser-desorbed ions are also of greater concern for high-mass ions. We are currently investigating the parameters influencing the mass measurement accuracy of high-mass ions. ACKNOWLEDGMENT This research was supported by grants from the National Institute of General Medical Sciences, the U.S.Department of Energy, Division of Chemical Sciences, Office of Basic Energy Sciences. We thank Dr. Gary R. Kinsel and Dr. Kermit K. Murray for their valuable discussions. Received for review December 8, 1993. Accepted February 18, 1994.' Abstract published in Advance ACS Abstracts. April 1, 1994.

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