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Anal. Chem. 1003, 65, 312-316
Band-Pass Kinetic Energy Filter for Postionization Separation of Proteins by Electrospray Ionization/Fourier Transform Ion Cyclotron Resonance Mass Spectrometry S. A. Hofstadler, 5.C. Beu, and D. A. Laude, Jr.* Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas 78712
A postlonization technique Is developed for separatlng electrosprayed protein mlxtures that have been introduced to a FourkrtransformIon cyclotron resonancemassspectrometer (FTICR). Electrosprayed Ions are selectively trapped In the FTICR trapped Ion cell on the bask of differences in kinetic energies as they emerge from the supersonlc expansion of the ESI Interface. Factors determlning the reiatlve order of trapplng at Increasing accumuiatlon potentlais In the range 1-10 V at the cell are the potentlal applied to the skimmer cone and ion veioclty In the expanslon. A model for optlmum trapping potentlal Is developed and found to depend primarily on ma88 to charge ratio wlthin the electrospray charge envelope but a b on protellFspeclflcfeatures lndudlngvelocity silp in the expanslon and effectlve potentlai experlencedupon exiting the sklmmer cone. For a three component mixture of cytochrome c, bovlne albumin, and chlcken egg whlte lysozyme, optimum accumulatlon potentlals of 3.5, 5.0, and 6.5 V, respectively, are employed to generate ESIIFTICR spectra of the lndlviduai protelns.
Electrospray ionization (ESI) has proven to be an efficient method for the production of multiply charged, intact molecular ions from a variety of analyte molecules.1-5 Since the first successful coupling of ESI with a modern mass spectrometer by Fenn and eo-workers in 1984: ESI/MS has been used to analyze a wide range of compounds including polymers, transition-metal complexes,7 oligonucleotides,8 quaternary ammonium salts: and most commonly, peptides and proteins10Jl with molecular weights extending beyond 200 000.12 Of increasing importance to ESI/MS is the practical application to samples of unknown concentration in solutions with a large number of contaminants and impurities. Obtaining quality ESI mass spectra from such complex samples (1)Fenn, J . B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9, 37-70. (2) Smith, R. D.; Loo, J. A.; Edmonds, C. G.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1990,62, 882-899. (3) McLuckey, S. A.; Van Berkel, G. J.; Glish, G. L. J . Am. Chem. SOC. 1990,112, 5668-5670. (4) Calaycay, J.; Rusnak, M.; Shiveley, J . E. Anal. Biochem.1991,192, 23-31. (5) Mann, M.; Meng, C. K.; Fenn, J. B. Anal. Chem. 1989, 61, 17021708. (6) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4451-4459. (7) Katta, V.; Chowdhury, S. K.; Chait, B. T. J . Am. Chem. SOC.1990, 112, 5348-5349. ( 8 ) Bruins, A. P.; Covey, T. R.; Henion, J . D. Anal. Chem. 1987, 59, 2642-2646. (9) Loo, J . A.; Udseth, H. R.; Smith, R. D. Biomed. Enuiron. Mass Spectrom. 1988, 17, 411-414. (10) Katta, V.; Chowdhury, S. K.; Chait, B. T. J . Am. Chem. SOC.1990, 112, 5348-5349. (11)Meng, C. K.; Fenn, J. B. Org. Mass Spectrom. 1991,26,542-549. (12) Feng, R.; Bouthiller, F.; Konishi, Y.; Cygler, M. Proceedings of the 39th ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, T N , 1991; pp 1159-1160.
0003-2700/93/0385-03 12$04.00/0
is difficult without extensive sample pretreatment, and consequently, sample purification is a common step in ESII MS analysis. Sample purification is well established among protein chemists who have developed a variety of separation procedures and highly sensitive assays.13-15 More recently, as mass spectrometry has been applied to protein analysis, sample preparation has increasingly involved the on-line coupling of separation techniques to the mass spectrometer to improve performance and simplify spectral interpretation. This is especially true for ESI/MS which is potentially more compatible with column chromatography. Sample cleanup techniques for ESI can be divided into two categories, those that take place prior to ionization and those that take place following ionization but prior to mass analysis. Most commonly, the bioseparations community employs preionization techniques such as capillary zone electrophoresis (CZE)16J7 and high-performance liquid chromatography (HPLC). Successful interfaces between HPLC and ESI/MS have appeared in the last several years.1s23 Alternatively, Smith and coworkers have pioneered the couplingof CZE with ESI/MS.24-29 In contrast to the above mentioned preionization separation techniques, it is possible in some cases to separatethe analyte after electrospray ionization. This type of postionization separation can be traced to the original electrospray experiments performed by Dole and co-workers30 in which a retarding grid was placed in front of a Faraday cage ion collector. A molecular beam consisting of N2, solvent (13)Hancock, W. S. LC-GC 1992,10, 95-99. (14) Diamandis, E. P.; Christopoulos, T. K. Anal. Chem. 1990, 62, 1149A-1157A. (15)Briggs, J.; Panfili, P. R. Anal. Chem. 1991, 63, 850-859. (16)Smith, R. D.; Olivares, J. A.; Nguyen, N. T.; Udseth, H. R. Anal. Chem. 1988,60, 436-441. (17) Jorgenson, J. W.; Lukacs, K. D. Science 1983,222, 266-272. (18)Hiraoka, K.; Kudaka, I. Rapid Commun. Mass. Spectrom. 1990, 4 , 517-524. (19)Sakairi, M.; Yergey, A. L. Rapid Comrnun. Mass. Spectrom. 1991, 5, 354-356. (20) Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J . B. Anal. Chem. 1985,57, 675-679. (21) Henion, J. D.; Duffin, K.; Wachs, T. Presented at the 38th ASMS Conference on Mass Spectrometry and Allied Topics, Tucson, AZ, 1990. (22) Huddleston, M. J.; Bean, M. F.; Barr, J. R.; Carr, S. A. Presented a t the 38th ASMS Conference on Mass spectrometry and Allied Topics, Tucson, AZ, 1990. (23) Simons,D. S.;Colby,B. N.;Evans,C.A., Jr.Int.J.MassSpectrom. Ion Phys. 1974, 15, 291. (24) Smith, R. D.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1988, 60, 1948. (25) Olivares, J. A.; Nguyen, N. T.; Yonker, C. R.; Smith, R. D. Anal. Chem. 1987,59, 1286-1290. (26) Smith, R. D.; Loo, J. A.; Baringa, C. J.; Edmonds, C. G.; Udseth, H. R. J. Chrornatogr. 1989, 480, 211-219. (27) Loo, J . A.; Udseth, H. R.; Smith, R. D. Anal. Biochem. 1989,179, 404-4 14. (28) Edmonds, C. G.; Loo, J . A,; Baringa, C. J.; Udseth, H. R.; Smith, R. D. J . Chromatogr. 1989, 474, 21-29. (29) Smith, R. D.; Loo, J. A.; Baringa, C. J.; Udseth, H. R. Anal. Chem. 1988,60, 1948-1952. (30) Dole, M.; Mack, L. L.; Hines, R. L.; Mobley, R. C.; Ferguson, L. D.; Alice, M. B. J . Chem. Phys. 1968, 49, 2240. 0 1993 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 65. NO. 3,FEBRUARY 1. 1993 313
I
I
''
Diffusion
RoughPump. To 13 Us Rough Pump
Flgure 1. Multiple wncemric vacuum chamber that allows Ion formatlonand detection to take place wkhln the bore of the 3-T superconducting and Torr In the analyzer trapped Ion cell with a neutral magnet. Pressures are at atmosphere In the ESI source and variable between background gas added to exploit collislonaI coollng In the Cell.
molecules, and electrosprayed polystyrene ions was sampled by a skimmer cone resulting in an isovelocity stream of particles. More recently, Feng and c o - ~ o r k e r sobserved ~~ that for the electrospray of contaminated solutions, proteins typically have significantly higher kinetic energy than nonprotein components. In the case of a papain (MW = 23 422) solution which was contaminated witb poly(propy1 glycol) (MW = 2W2400), the papain ions (+14 to +19) were found to exit the ESI source witb a kinetic energy approximately 11eV per charge greater than that of the poly(propy1 glycol) ions (+1 to +2). Due to this significant kinetic energy difference it was feasible to bias the quadrupoles a t an appropriate potential to serve as a retarding grid, allowing only the higher kinetic energy papain ions to enter the quadrupole for mass analysis. Additionally, this technique was used t o remove degradation fragments weighing 13400 kDa, 23 600, and 47 300 from a sample of anti-(al-acid glycoprotein), a 150IgG class antihody.31 From these findings Feng concluded that concomitant nonprotein and peptide ions have less kinetic energy than the heavier protein ions and that this kinetic energy difference can be used to clean up mass spectra of proteins. Thus, one way in which this postionization separation might be described is as a highpass kinetic energy filter in which the undesirable contaminants possess a relatively low kinetic energy and are filtered from the mas8 analyzer. To he presented here is a postelectrospray ionization separation technique used in conjunction with high magnetic field ESI/FTICR that acts as a hand-pass kinetic energy filter in thatbothunwantdlow-andhigh-energyionsarediscarded. The resultsto be presented follow fromprevious ohservations of a strong correlation between the kinetic energy of the electrospray ion beam and the potential a t which ions are mostefficientlytrappedforsubsequent detection byFTICR.32 Specifically, the ion trapping efficiency is optimized when the kinetic energy of the ion heam matches the potential applied to the trap plates of the trapped ion cell. Thus, only anarrow sliceor band-pass of ions that possess the appropriate kinetic energy are trapped because low kinetic energy ions have insufficient kinetic energy to penetrate the trapping well. Similarly,ions that poasess kinetic energies greater than the trap plate potential possess sufficient kinetic energy to exittbe trappingwell. Given thatproteinsand contaminants of different molecular weights might he expected to exhibit different kineticenergy profiles, it was decided to investigate the degree of selectivity possible when mixtures were analyzed by ESI/FTICR. Examples to be presented include resolution of a three-component mixture of cytochrome c, chicken egg white lysozyme, and bovine albumin by selective trapping. A model is developed which predicts that relative trapping order is primarily dependent on the mass-to-charge ratio of the
electrosprayed ion hut also on velocity slip in the expansion and on the effective field experienced by ions upon exiting the expansion. EXPERIMENTAL SECTION Instrumentation. All experiments were performed on a FTICR assembled with components that constitute the Extrel FTMS-2000 including a 3.0-T superconducting magnet, Nicolet 1200 computer, cell controller,high-power excitation amplifier, and differentiallypumped dual-cellassembly. The electrospray interface was previously described3*so only a brief description is given here. The electrosprayion source is modeled after that introduced by Chait and co-workersa in which a heated stainless steel capillary is used to desolvate the charged droplets formed by electrospray. A pressure differential is maintained across this positively biaseddesolvating capillary bya high-speed rough pump; pressures in the rough pumped chamber are typically a few Torr. This pressure differential sets up a supersonic expansion which is sampled by a biased skimmer cone. After crossing multiple stages of differential pumping the ion heam is mass analyzed. In order to accommodate the high-pressure requirements for ion formation and the low-pressure requirement for FTICR ion detection, a vacuum chamber was constructed which consists of four differentiallypumped multiple concentric tubes, as shown in Figure 1. The innermost tube of this seriesconsists of a hollow 3/~-in. stainless steel probe which houses the electrospraycavity and heateddesolvationcapillary.this prohe-mountedESIsource is inserted into the vacuum chamber through a Vr-in. solids probe inlettoalign witha330-pm4.d. copper skimmer cone. Thevarioua conductance limits and high-speed pumps allow operating pressures in the l~w-lO-~-Tarr and mid-lO"-Torr range in the analyzer and source sides of the dual cell, respectively. A more recent spectrometer configuration utilizes a 200-pm-i.d. copper skimmer cone that reduces analyzerpressures tothe low-lW-Torr range. Electrospray Parameters. Proteinsolutions were prepared with analyte concentrations of 3-10 pmol/pL in a 68302 MeOH HpOHoAc matrix. All proteins were obtained from Sigma Chemical and used without additional purification. Solutions were pumped to the electrospraychamber at 4 pLlmin through a 22-gauge Teflon tube hy an Isco Model SFC-500 microflow syringe pump. Electrospray ionization was performed by applyingapotentialof 3.7 k V t o the 100-jcm-i.d. blunt-ended syringe needle. The syringe needle was positioned 6 mm from the desolvating capillarywhich was biased to 330 V and resistively heated to -150 "C by an applied current of 2.2 A. The other end of the desolvating capillary was positioned 4.5 mm from the (31) Feng, R.; Konishi, Y. Presented at the 12th International Mass Spectrometry Conference,Amsterdam,The Netherlands, August 26-30, lWl
(321 Hofatadler, S. A,; Laude, D. A., Jr. J. Am. Sac. Moss Spectrom. 1992,3,615-623. (33) Chowdhury, S. K.; Katta, V.; Chait, B. T. Rapid Commun. M-8 Speetrom. 1990, I , 91-97.
314
ANALYTICAL CHEMISTRY, VOL. 65, NO. 3,FEBRUARY 1, 1993 Vskim = 5 v
1124 1
P
1
Vskim = 7 . W
Vskim = 22SV
4 2
0 0
2
4
6
8
10
AccumulationPotential(Volts) Figure 2. Ion accumulation profiles for the charge envelope of cytochrome cobtained at increaslng sklmmer cone potentlal. For this particular set of profiles, pressures in the analyzer chamber were Torr, and ions were found to be most efficlently trapped when the accumulatlon potential was approximately one-third of the skimmer
cone potential. skimmercone which was biased at +5 to +30 V in order to control the kinetic energy of the ion beam. Ion Trapping and Detection. A standard FTICR pulse sequence was used for these experiments. An appropriately selected trapping potential maintained on both source and analyzer trap plates allowed ions to accumulate in the trapped ion cell during a several hundred millisecond period as the cell filled to capacity. During the subsequent thermalization delay a potential of 9.75 V was applied to a conductancelimit to prevent additionalions fromenteringthe cell while the source and analyzer plates are maintained at the accumulation potential. The trap plate potential was then lowered to 1 V during a 0-100-kHzlinear excitation sweep and subsequent broad-band detection of 16K data points. Transients were processed by a single zero fill, baseline correction, sine-bell apodization, and magnitude mode Fourier transform.
RESULTS AND DISCUSSION Trapping Externally Generated Ions. Two approaches have been used to trap ions injected into the FTICR trapped ion cell from an external ion source. In gated trapping one or both of the trap plates are momentarily set to ground potential to allow ions into the cell; upon reapplication of the trapping potential, ions are retained if the sum of the kinetic and potential energies of these ions does not exceed the depth of the trapping well. Although a theoretical evaluation of this trapping procedure indicates that ion loss due to nonadiabatic potential changes can be minimized if the proper a significant disadvantage is that trapping scheme is the duty cycle for the gated trapping experiment is incompatible with continuous ionization sources. In contrast, accumulated trapping requires that the trap potential be maintained during the injection process a t a static potential that is determined by ion kinetic energy. Ion trapping occurs as long as kinetic energy and trap potential are matched, which makes accumulated trapping well suited for continuous sources such as electrospray ionization. The duty cycle for ion injection is greatly increased and large gains in sensitivity may be possible. Thus accumulated trapping is used exclusively in these experiments. For the ESIIFTICR instrument we have constructed, the kinetic energy of the ion beam is best regulated by the potential applied to the skimmer cone. Presented in Figure 2 are ion accumulation profiles which measure ion abundance at various trapping potentials for the +14 charge state of cytochrome c obtained at increasing skimmer cone potentials. As will be explained, ions are most efficiently trapped when the accumulation potential under this set of experimental parameters (34) Hofstadler, S. A.; Lande, D. A., Jr. Int. J. Mass Spectrom. Ion Processes 1990, 101, 67-18.
4.5 5 5.5 6 6.5 I accumulation potential (V) Figure 3. Normallzed FTICR magnltudes for the +14 (01+15 , (A), and +16(O)chargestatesofcytochromecacqulredatafixedsklmmer potential of 15 V as the accumulation potentlal is increased.
3.5
4
approximates the effective electric field between the skimmer and the conductance limit. This is because the electric field along with ion velocity emerging from the expansion determines the kinetic energy of the electrospray ions at the cell. In general, the effective field increases as the pressure drop across the expansion increases. In addition, the effective field depends on the velocity of the protein upon exiting the skimmer. On the basis of a model to be developed later in the paper, estimated values for effective potential were 10% to 30% of the applied potential for the larger 330 pm4.d. skimmer orifice but approached 100% for the smaller 200Km4.d. orifice across which a substantially larger pressure drop was achieved. The data in Figure 3 illustrate the charge dependence of the accumulation process. Accumulation profiles for the +14, +15, and +16 charge states of cytochrome c indicate that trapping at higher accumulation potential favors higher rnlz ions. This charge shifting phenomenon makes it possible to control, to some extent, which charge states are trapped. The extent of charge envelopeshifting with accumulation potential is protein specific and therefore may prove useful as a probe of gas-phase protein structure. For example, the observed charge envelope of chicken egg white lysozyme is only slightly influenced by the accumulation potential and contrasts with turkey conalbumin which exhibits radical shifts in the observed charge envelope. Turkey conalbumin peaks corresponding to mlz values as high as 4862 are observed if a 10-Vaccumulation potential is utilized. It should be mentioned that the enormous shift in the turkey conalbumin charge envelope is unique among the proteins investigated. Variable accumulation potential studies indicate that the pressure in the analyzer cell is an important factor in determining the extent to which the charge envelope shift occurs. This suggests that reactive collisions between the more highly charged ions and background neutrals may be responsible although some type of charge-dependent ion loss from the cell also may be occurring. Evidence that the charge shifting is collision-based is presented in Figure 4 where ESI/ FTICR spectra are acquired for two proteins following ion accumulation at 3 X Torr with no delay and following a 5-5 thermalization delay under otherwise identical conditions. In Figure 4a, cytochrome c is trapped and detected with a charge envelope in the mlz 688-1031 range but after a 5-5thermalization delay a shift to the mlz 728-1374 range is observed. In Figure 4b, bovine albumin is initially trapped with a charge envelope corresponding to mlz 1200-1800 but after a 5-5 thermalization delay the observed charge envelope shifts to rnlz 1600-5000. Accumulation Profiles of Different Proteins. Most pertinent to the idea of postionization separation is the
ANALYTICAL CHEMISTRY, VOL. 85, NO. 3, FEBRUARY 1, 1993
lodo
315
1560
mh
'4
"wl^
m
m/z
m
Flgum 8. Cytochrome-c and chicken egg white lysozyme mixture used in Figure 6 separated at increasing skimmer potential for a fixed accumulation potential of 5.0 V.
4Ooo
2000 d Z
Flgure 4. Extent of postiinizationcharge shifting shown to be protein dependent with spectra acquired following a 500-m~injection event and no thermalization delay in the top spectrumor an equivalentInjection event and a 5-s thermalization delay in the bottom spectrum for (a) cytochrome c and (b) bovine albumin.
Y 1500 , ?
*-
m/z
Flgwo7. Equimdarthree-componentmixtureof cytochrome c.bovine albumin, and chicken egg white lysozyme separated using a fixed skimmer potential of 14 V at variable accumulation potentials.
500
1000
1500
2000
d Z
Figure 5. Separation of a two-component mixture of cytochrome c (3 pmol/pL) and chicken egg white lysozyme (7 pmol/pL). Spectra are acquired with a fixed skimmer cone potential of 12.5 V as the accumulation potential is increased.
possibility of distinguishingdifferent proteins directed to the cell under identical conditions. The dependence of accumulation potential on mass-to-charge ratio suggests that this should be possible if the charge envelopes from different components in a mixture appear in different regions of the mass spectrum. Accumulation profiles of individual cytochrome c and bovine albumin were acquired under identical conditions. These accumulationprofiles were superimposed, showing that the optimum accumulation potential for cytochrome c is approximately 1.75 V lower than the optimum accumulation potential for the bovine albumin and that sufficient resolution should exist to separate a mixture of the two proteins. Figure 5 demonstrates a simple two-componentmixture of cytochrome c and chicken egg white lysozyme at concentrations of 3 and 7 pmoVfiL, respectively. Spectra are acquired with a fixed skimmer cone potential of 12.5 V as the accumulation potential is systematically increased. At an accumulation potential of around 4 V, only cytochrome c ions are accumulated in the trapped ion cell, while near 6 V, only chicken egg white lysozyme ions are trapped. As an
alternative method for selective trapping of proteins, the kinetic energy of the ions rather than the accumulation potential can be altered. This can be done by varying the skimmer cone potential while using fixed accumulation potential. Figure 6 illustrates such a separation in which the accumulation potential is fixed at 5 V and the skimmer cone potential is varied from 5.0 to 17.5 V. As was the case in Figure 5, pure spectra of each component are obtained, this time at 10.0- and 15.0-V skimmer potentials. Although the data in Figures 5 and 6 indicate protein separation is based primarily on differences in mlz, additional effects apparently facilitate the separation of proteins with overlapping charge states but different masses. This is important for extending the general utility of the technique since eledroeprayionization necessarily compresses the usable mlz range for detection. Presented in Figure 7 is a threecomponent separation of cytochrome e, chicken egg white lysozyme, and bovine albumin. As was previously demonstrated, cytochrome c can be isolated from bovine albumin and chicken egg white lysozyme at lower accumulation potentials, but surprisingly, chicken egg white lysozyme at a 7.0-Vaccumulation potential is isolated from the lower energy bovine albumin despite identical mlz ranges. These data suggest that for the case of proteins with overlapping rnlz ranges, heavier mass proteins are apparently favored for retention at a lower accumulation potential. As will be described, we believe this phenomenon is related to velocity slip in the supersonic expansion. Model for Kinetic Energy Filtering. Several empirical observations were used to create a model for optimum trapping potential for a specific protein as well as elution order. From the data it is evident that mlz, rather than mass, is the most important parameter for elution order. In addition the potential applied to the skimmer was found to have a strong influence on Vtrapbut the potential applied to the capillary
316
ANALYTICAL CHEMISTRY, VOL. 65, NO. 3, FEBRUARY 1, 1993
cytochrome c vel. = 900 4 s
turkey conalbumin curve in Figure 8, conditions included a V&im of 15 V, a skimmer-capillary distance of 3 mm, and an analyzer pressure of Torr. Using eq 4,the velocity out of the expansion is calculated a t 599 rnls and the effective Vskim extrapolated to they intercept is 3.2 v. Also presented in Figure 8 is a curve for cytochrome c generated for an 11-V skimmer, 10-mm capillary-skimmer distance, and 10-5-Torr pressure. Under these conditions the velocity is calculated at 900 rnls and the effective skimmer potential is 0.85 V.
turkey conalbumin vel. = 599 4 s V,y= 3.2 V
c
0
400
800
1200
1600
2000
m/Z
Flgurr 8. Fit of eq 4 to the optlmum accumulationpotential for each charge state of cytochrome c and turkey conalbumin. Predicted ion velocities and effective potentials for the two proteins are presented with the plots.
had no effect. Also of interest is the observation that the effective potential experienced by an ion is approximated by the ratio of Vtrapto Vskim and is found to increase with decreasing system pressure. This change in the proportionality constant relating V&bto Vtrapis likely due to the smaller skimmer diameter which reduces the analyzer pressure while increasing the expansion velocity. The distance between capillary and skimmer is also of critical importance, with Vtrapfor optimum signal intensity decreasing sharply with increasing distance between the capillary and skimmer. In general, a capillary-skimmer distance was chosen which provided the lowest pressure in the trapped ion cell and coincidentally produced ion kinetic energies per unit charge a t the cell of only a few electronvolts. It is not surprising then that this optimum distance for pressure also yielded the highest trapping efficiency a t the trapped ion cell. A simple model is now constructed to estimate Vtrap. It is assumed that the kinetic energy of the ESI ions must match Vtrapfor the ions to be retained. The two major contributions to the kinetic energy are the velocity component achieved in the supersonic expansion and the electric field component derived from the potential drop between the Vsbmand the grounded conductance limit. These are expressed as the fiist and second terms, respectively, in eq 1
where m is the mass in kilograms, u is the expansion velocity in meters per second, Kslipis a correction factor for velocity slip in the expansion, q is the ion charge in Coulombs, Vskim is the skimmer potential in volts, and Keffisa correction factor for the effective field experienced by the ions. The kinetic energy is expressed in electronvolts in eq 2
KE,, = Kslipmu2/2e+ KefflVskim/e (2) by dividing by the elementary charge, e. The optimum trapping potential is then obtained in eq 3 by dividing eq 2 by z , the number of elementary charges on the ion. (3) Vtrap= Kslipmv212ez+ KefflVskim/ez Because ez = q and by converting into Daltons, A, eq 3 simplifies to
Vtrap= ~slip(u212Ae)(mlz) + KeffVBkim
(4)
Note that eq 4 exhibits the demonstrated relationship between Vtrapand mlz within the electrospray ion packet. Presented in Figure 8 are illustrative examples of this effect. In all cases, Vtrapwas assigned on the basis of the maximum signal magnitude generated for each charge state. For the
The profiles in Figure 8 indicate that, by variation of the parameters that determine expansion velocities (Le., pressure and orifice size) and effective electric field (vskim), different values for Vtrapcan be generated. Evidence from Figure 7 suggests that there is apparently sufficient variation in Ksfip and K,, from protein to protein to separate even those with similar mlz distributions. Thus, for example, velocity slip arguments must be considered to explain the separation of bovine albumin from chicken egg white lysozyme. Although little is known about the relative magnitude of velocity slip for very large molecules, it is likely to increase with the size of the protein. Thus bovine albumin will experience the largest velocity slip and consequently will be captured at a smaller Vtrapthan the smaller chicken egg white lysozyme. Finally, according to eq 4 it should be possible to reduce Vskh and effectively capture ions at a much lower trap potential. It was found that this could be accomplished in practice by employing the smaller skimmer which yielded pressures in the analyzer chamber in the 10-8-Torr range. With this skimmer Keffapproached unity, Vtrap approached Vskim, and realistic Vskimvaluesof 1-2 V could be used. Under these conditions the separation voltage range for Vtrap, which was 3-4 V for separations in Figures 5 and 7, was reduced to less than 1 V, as increasingly the velocity component of eq 4 governed the magnitude of Vtrap. With the compression of the working Vtraprange, it might be expected that separation performance would deteriorate. In fact, as the data in Figure 2 indicate, peak widths decreased proportionally with the decrease in Vtrap. Thus in the new separation of cytochrome c chicken from egg white lysozyme at lower pressure, optimum accumulation potential widths for the two proteins were 1.21.4 V and 1.5-1.7 V, respectively. Future efforta to improve upon the band-pass kinetic energy filter for selecting electrospray ions for FTICR detection will require a better understanding of phenomena that determine ion kinetic energy so that appropriate instrumentation modifications can be made to enhance separation efficiency. Toward that end, better control of the supersonic expansion will be attempted to obtain a more uniform beam velocity of protein ions. Although it is unlikely that the postionization band-pass filter just described will ever achieve sufficient resolution to compete with chromatographic methods in the separation of complex mixtures, it does offer an added dimensionto spectral analysis of electrospray ions by removing contaminating ions associated with unresolved mixture components and matrix materials in the spray.
ACKNOWLEDGMENT This work was supported by the Arnold and Mabel Beckman Foundation, the Texas Advanced Research and Technology Program, and the National Science Foundation. RECEIVED for review June 24, 1992. Accepted November 3, 1992.
