Anal. Chem. 1994,66, 126-133
Reflecting Time-of-Flight Mass Spectrometer with an Electrospray Ion Source and Orthogonal Extraction Anatoli N. Verentchikov, Werner Ens, and Kenneth 0. Standlng' Department of Physics, University of Manitoba, Winnbeg, Manitoba, Canada R3T 2N2 An electrospray source has been coupled to a reflecting timeof-flight mass spectrometer. The ions enter as a continuous beam in a direction perpendicular to the spectrometeraxis and are formed into short bursts of ion0 with velocities parallel to the axis by electrical pulses applied to injection electrodes. The instrument may be operated either in the linear mode, with ions detected behindthe electra&atic mirror, or in the reflecting mode, witb ions detected after reflection. In the latter case the time resolution is I 8 ns for m / z -600, enabling observation of individual isotopic peaks for masses up to about 4000 u. The sensitivity is adequate to enable measurement of mass spectra for 10 fmol of cytochrome c (- 12 000 u). The spectrometer does not limit the range of m / z values, and ions have been observed up to m / z --6ooo. With proper adjustment of pH and other conditions in the source, the ionization is very soft, enablinginjection of weakly bound complexes; these have been accelerated and measured in the spectrometer without observable fragmentation. Spray ionization of biopolymers, pioneered by M. Dole et a1.l and first coupled with quadrupolez-" and magnet sector mass spectrometers?is now recognizedas a powerful analytical method.6~~ It has become particularly valuable in biochemistry since the discovery that electrospray (ES) produces multicharged ions from large biopolymers with a mass to charge ratio ( m / z )around 1000,suitable for inexpensivequadrupole filters and easily detectable by conventional electron multipliers.* Electrospray sources have also been coupled to time-offlight (TOF) mass spectrometer^."^ In principle such (1) Dole, M.; Mack, L. L.; Hines, R. L.; Mobley, R. C.; Ferguson, L. D.; Alice,
M. B. Chem. Phys. 1968,49,2240. (2) Iribame, J. V.;Dzicdzic, P. J.; Thomson, 9. A. Inf. J . Mass Spcctrom. Ion Phys. 1983, 50, 33 1. (3) Yamashita, M.; Fenn, J. B. J . Chem. Phys. 1984, 88, 4451. (4) Kambara, H.Anal. Chem. 1982,51, 143. (5) AlekPandrw,M. L.;Verentchikov,A.N.;Gall,L.N.;Krasnov,N. V.;Masalov, S.A.; Nikolayev, V. I.; Shkurov,V. A,; Scherbakov, A. P. Research Report No. 02830037141; Institute of Analytical Instrumentation of the USSR Academy of Sciencc: Leningrad, 1982. Aleksandrov, M. L.; Gall, L. N.; Krasnov, N. V.; Nikolaycv, V. I.; Pavlenko, V.A.; Shkurov, V. A. Dokl. Akad. Nauk SSSR 1984,277, 379; Dokl. Phys. Chem. 1985, 277, 572. (6) Fenn, J. B.;Mann, M.;Meng,C. K.; Wong,S. F. MassSpecfram. Rev. 1990, 9, 37. Mann, M.; Fenn, J. B. Mass Specrromefry, Clinical and Biomedical Applicafiuns, Vol. I; Plenum: New York, 1992; pp 1-35. (7) Smith, R. D.; Loo, J. A.; Loo, R. R. 6.; Busman, M.;Udacth, H. R. Mass Specfrom.Rev. 1991,10,359. Smith, R.D.;Loo, J.A.;Edmonds,C.G. Mass Specfromefry,Clinical and Biomedical Applications, Vol. I; Plenum: New York, 1992; p 37. (8) Meng, C. K.; Mann, M.; Fenn, J. B. Z . Phys. 1988, DIO,361. (9) Dodonov, A. F.; Chemushcvich, I. V.; Dodonova, T. F.;Raznikw, V. V.; Tal'rozc, V. L. USSR Patent No. 1681340A1, Feb 1987. (10) Dodonov, A. F.; Chemushcvich, I. V.; Laiko, V. V. International Mass Spectrometry Conference, Amsterdam, August 1991; Extended Abstracts, p 153. Dodonov. A. F.; Chernushevich, I. V.; Laiko, V. V. In Time-of-Flight Mass Spectromefry;Cotter, R. J., Ed.; ACS S y m p i u m Series; American Chemical Society: Washington, DC, in press.
126 AnaWcal Chemistry, Vol. 66, No. 1, January 1, 1994
instruments have a number of useful properties. The most obvious one is that a TOF spectrometerdoes not by itself limit the observable mass-to-charge ( m / z )range, although there may be limitationsin the source or detector. Fenn et a1.6 have presented arguments that the range of m/znormally produced by ES sources is restricted to values 12000, for which quadrupole spectrometersare suitable. However, it is difficult even to checkthis prediction experimentallyin most quadrupole instruments because of their rapid decrease in efficiency as m/z increases. Clearly there are cases where, it would be useful to examine a higher m/z range.16J7 A second advantage of time-of-flight techniques is their ability to record all ions striking the detector without scanning. This can lead to higher accuracy in mass determination, since ions of all masses are measured at the same time. It also enables very fast response, useful when measuring chromatographic outputs. However, perhaps the most important aspect of this feature is the increase in efficiency that it provides when a pulsed ion source is used. On the other hand, coupling a continuous ion source, such as electrospray, to a TOF spectrometer may involve large losses in intensity. Fortunately TOF instruments can tolerate a relatively large spatial or velocity spread in a plane perpendicular to the spectrometeraxis (the z axis), as witnessed by the large sources ( I1-cm diameter) typically used in fission fragment desorption. This tolerance has been exploited by injecting electrospray ions into the TOF instrument in a J~J5 direction perpendicular to the z a x i ~ . ~ - ~ ~Orthogonal injection has also been used with other types of ion source; see, for example, refs 18-22. This geometry provides a highefficiency interface for transferring ions from a continuous beam to a pulsed mode. A second advantage is the small velocity spread in the z direction that is usually observed, making high resolution easier to obtain. The existing ES/TOF instruments have already achieved high sensitivity (in the femtomole range14) and reasonable ( 1 1) Chcrnushevich,I. V.Ph.D. Thesis, Instituteof EnergeticProblemsof Chemical
Physics, Chernogolovka, 1991. (12) Verentchikov, A. N.Ph.D. Thesis, Institute of Analytical Instrumentation of the USSR Academy of Science, Leningrad, 1990. (13) Boyle, J. G.; Whitehouse, C. M.; Fenn, J. B. Rapid Commun.Mass Spcctrom. 1991, 5, 400. (14) Boyle, J. G.; Whitehouse, C. M. Anal. Chem. 1992, 64, 2084. (15) Zhao, J.; Zhu, J.; Lubman, D. M. Anal. Chem. 1992.64, 1426. (16) Feng, R.; Konishi, Y. Anal. Chem. 1992,64,2090. (17) Mirza, U. A.; Cohen, S.L.; Chait, B. T. Anal. Chem. 1993.65, 1. (18) Gerhard, P.; Loffler, S.; Homann, K. H. Chem. Phys. Lett. 1987, 137, 306. (19) Olthoff, J. K.; Lys, I. A.; Cotter,R. J. Rapid Commun. MassSpectram. 1988, 2,171. Emary, W. B.; Lys, I.; Cotter, R. J.; Simpson, R.; Hoffman, A. Anal. Chem. 1990,62, 1319. (20) Bergmann, T.; Martin, T. P.; Schaber, H. Reu. Scl. Insfrum. 1989,60,792. (21) Dawson, J. H. J.; Guilhaus, M. Rapid Commun. Mass Specfrom. 1989, 3, 155. (22) Sin, C. H.; Lee, E. D.; Lee, M. L. Anal. Chem. 1991, 63, 2897.
0003-27OOl94lO368-0126$O4.50/0
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Flguro 1. Schematlc diagramof the reflectingTOF mass spectrometer (TOF 111) with orthogonally injected ES ions.
resolution for light ions ( M / A M ~ H = M 20001OJ1 and = 1000L4),but the resolution is much poorer for large masses. For example, the resolution for bovine insulin (5733 u) has been measured as -300,9112 and the spectra reported for cytochrome c (12 000 u) seem to correspond to a value in the same range.'" This limited resolution is a serious impediment when examining large biomolecules. We have recently coupled a reflecting time-of-flight spectrometerwith an electrospray ion sourceusing orthogonal ion injection; data were acquired with a time-to-digital converter using single-ion ~ u n t i n g . 2Our ~ early evaluation of the properties of this instrument is reported here.
EXPERIMENTAL METHOD The instrument (TOF 111), shown in Figure 1, consists of a reflecting time-of-flight mass spectrometer, with its axis vertical, and an electrospray ion source floated at the acceleration potential. The source continuously injects a slow (- 10 eV per charge) horizontal ion beam into the spectrometer, where it is formed into vertically accelerated ion packages by an orthogonal injection system. Time-of-Flight Mass Spectrometer(TOFIII). Apart from the ion source, the instrument is almost identical to our spectrometer TOF II,24 used for measurements in secondary ion mass spectrometry and matrix-assisted laser desorption. The vertical stainless steel vacuum chamber has a diameter of 25 cm and a length =1.2 m. It contains a single-stage electrostatic mirror similar to the one in TOF II?4 in which resolving powers M/AM-HM up to 13 000 have been obtained for alkali halide ions and up to 5000 for organic ions of mass C-3000 u, where the secondary ions were produced by pulsed Cs+ ion b~mbardment.~"The mirror consists of 30 rings; it has an inner diameter of 12 cm and is 33 cm long. The diameter of the entrance window of the mirror is 8 cm, somewhat larger than in TOF 11,because of
--
(23) Verentchikov, A.; EM, W.; Standing, K. 0.Proceedings of the 41st ASMS Conference on Mass Spectrometry and Allied Topica, 1993; p4. (24) Tang, X.; Bcavis, R.; EM, W.; Lafortune, F.; Schueler, B.; Standing, K. G. Int. J . Mass Spectrom. Ion Processes 1988, 85, 43. Tang, X.Ph.D. Thcsis, University of Manitoba, 1991.
Cr
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Fbwe 2. Schematic diagram of the ES ion source and the modulator for orthogonal injection: A, nozzle; Band C, first and second skimmer; D and E, defining slits; 1-4, extraction electrodes. the large dimensions of the ion source region. The potential of the entrance window is defined by a 90%transmission grid. The total flight time corresponds to an effective path length of -2.8 m. The orthogonal injection system and the detector for reflected ions are mounted symmetrically on the bottom flange of the chamber with 11 cm between centers. A detector for undeviated particles is mounted on the top flange. The spectrometer may be run either in the reflecting mode, with an electric field in the mirror, or in the linear mode, with the mirror voltage off. In the former case reflected ions are observed in the lower detector, while in the latter case all ions are observed in the upper detector. Each detector consists of two 40-mm-diameter microchannel plates in a chevron configuration. Some postacceleration is provided by setting the front of the detectors to -5 kV. In both cases, the detector signal passes through a 1-nF capacitor to a preamplifier and a constant-fraction discriminator with a 20-mV threshold. It is then registered by an Orsay time-to-digital converter (CTNM2) connected to an Atari TT ~omputer.~'The TDC can record up to 255 stops for each extraction pulse, but only one stop for a given m / z species. For high counting rates this could cause distortion. However, in the present experiment, the total counting rate is less than one ion per event (on average) so distortion of this kind is negligible. Electrospray Ion Source. The source constructed for the present instrument (Figure 2) is similar to the one designed by Aleksandrov et al. for a magnetic sector mas~spectrometer.~ Spraying is performed in a closed chamber, supplied with heated (70 "C) dry nitrogen through a gas sheath. A sample of the aerosol products produced enters the spectrometer through a three-stage differential pumping system. The inlet of the first stage is a nozzle with orifice diameter 0.5 mm; it is pumped by a 6 L/s mechanical pump to a pressure -5 Torr. The second stage follows a 0.15-mm-diameteraperture in a cone skimmer with a 3-mm-diameter flat tip, and is (25) Ens, W.; Standing, K. G.; Verentchikov, A. Proceedings of the International ConferenceonInstrumentationfor Timc-ofiFlight Mass Spectrometry;Blanar, G. J., Cotter, R.J., Eds.; M r o y Corp.: Chestnut Ridge, NY, 1993; p 137.
Analytical Chemistry, Vol. 66, No. 1, January 1, 1994
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evacuated by a 450L/s turbo pump to Torr. The third stage, entered through a 1.5- X 6-mm horizontal slit (cut in the flat tip of a second skimmer) is connected to the mass spectrometer chamber, which is pumped by a 350 L/s turbo pump to a pressure between 3 X and 5 X l e 7 Torr. The electrospray source as a whole is elevated to the dc acceleration potential, normally +4 kV. The relatively high pressure in the first stage inhibits discharges to the grounded mechanical pump; a 1-m length of plastic hose is sufficient to hold up to 10 kV at the 5 Torr of operating pressure. The region at gas pressures between 1 and 0.01 Torr, where discharges are likely, is screened from the electric field inside Torr, so the cone skimmer. The second stage runs at discharges to the grounded turbo pump are also inhibited. ES sources for quadrupole spectrometers commonly place the skimmer inside the Mach disk (stagnated shock wave) of the free jet issuing from the nozzle. The undisturbed core of the jet then diverges very slowly and is able to carry ions into the quadrupole without the assistance of a strong electric field. In contrast to this arrangement, our skimmer is placed beyond the Mach disk, 8 mm from the nozzle, so the stream of gas passing through the skimmer has a large divergence. As a result, ions passing through the skimmer suffer fewer collisions with the gas; such collisions add undesirable momentum in the vertical direction. The potentials applied to the source components gradually decrease from the spraying capillary to the final skimmer. Typical values are as follows: 3 -0-3.5 kV between the capillary and the nozzle to provide good conditions for spraying, as estimated by measurement of the spray current (100-200 nA); 50-350 V between the nozzle and the skimmer to provide controllable breakup of clusters and fragmentation of the ions; a few volts between the first and second skimmer to provide some focusing of the ion beam without significant broadening of the velocity distribution; 5-10 volts between the first skimmer and the injection electrodes. Possible fringing fields to parts of the system at ground potential are carefully screened by grids. Typical measured values of the ion currents are 100-200 nA for the total current of the sprayed aerosol, 10-20 nA through the nozzle, and 20-40 pA through the first skimmer. Orthogonal Injection. The third stage of the electrospray source is contained in an aluminum 5-in. cube, mounted on the bottom of the spectrometer so that the ion beam is perpendicular to thevertical axis of the spectrometer, as shown in Figure 1. As mentioned above, the ions are accelerated by 5-10 V between the final skimmer and a pair of additional horizontal slits (D and E) 2 mm wide and 20 mm apart, which are at the dc accelerating potential. These slits are intended to restrict the vertical beam dimension between the deflection electrodes to 54 mm and the vertical velocity spread to S1/s of the horizontal velocity spread. The ions are ejected into the spectrometer by a pulse applied to one of the horizontal deflection electrodes. Between extraction pulses both electrodes are at the dc accelerating potential (V, 4 kV), allowing the slow electrosprayed ions to fill the 50-mm-long storage gap. The ratio of the filling time (determined by the velocity of the electrosprayed ions) and the time between pulses (determined by the flight times of the ions through the spectrometer) determines the duty
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128 Ana&fical Chemistry, Vol. 66, No. 1, January 1, 1994
cycle. The velocity of the ions entering the injection gap depends on their mass because, in addition to the uniform velocity gained in the gas jet, their energy is increased by a constant amount (- 5 eV) per charge. The filling time is about 40 ps for ions with m / z 1000 and about 60ps for m / z 6000. Maximum flight time is about 250 ps, so the duty cycle is about 20%. The extraction pulse applied to the lower electrode has an amplitude of 400 V, a risetime of 10 ns, and a duration of 30 ps. The ion bunch is ejected through a 90% transmission grid (2) into the dc acceleration region and then across -4 kV through two more 90% transmission grids (3 and 4) into the Spectrometer. Electric field penetration through grid 1 can cause ions to leak out of the storage gap during the storage phase. To prevent this an intermediate grid (2) is held slightly above the acceleration potential between pulses; it is pulsed to 400 V below V, during extraction. The lengths of the pulsed and dc acceleration regions (6, 6,and 54mm) are chosen so as to give a uniform electric field over the three regions during the pulse. Under this condition ions originating at different vertical positions in the region between the plates are focused onto a horizontal plane after a free flight path twice as long as the acceleration region, i.e. a plane about 19 cm above their original position.26 This plane then serves as a virtual “object plane” for the spectrometer; the axial velocity spread of the ions in the object plane is corrected to first order by the electrostatic mirror.24 The ion beam has to be deflected by a few degrees after leaving the acceleration region so that the reflected ions will strike the detector. The angle of deflection is adjusted to take account of the geometry of the spectrometer and the initial horizontal component of ion velocity. Two pairs of deflection plates with symmetrical applied potentials and with field terminations on the ends were found to give adequate steering of the beam without significant deterioration of the resolution. The plates are 7 cm along the spectrometer axis, 5 cm wide, and 5 cm apart. For a vertical acceleration potential of 4 kV, the instrument acceptance corresponds to a range of horizontal energies between 2.5 and 10 eV per charge. Since light electrospray ions already have energy near this range, very little additional deflection is required; usually less than 70 V is applied to the deflection plates. Since part of the energy of the electrospray ions comes from the gasjet, the energy-and therefore the optimum deflection voltage-has a mass dependence. For ions with m / z above a few thousand, a deflection voltage between 100 and 300 V was used. This effect imposes some limitations on the range of m / z values that can be detected simultaneously without mass discrimination. Chemicals. Solutions of proteins with concentrations from le7to l e 5 M were prepared from deionized water (46%), reagent grade methanol (46%), and glacial acetic acid (4%), unless indicated otherwise. Peptides of masses GO00 u were dissolved at higher concentration, 10-4M in 90% methanol, 10% water, and 0.01% TFA. Thecompounds measured were obtained from Sigma Chemical Co. and were used without further purification.
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(26) Wiley, W. C.; McLaren, I. H. Reu. Sci. Znsrrum. 1955, 26, 1150.
ME M/z F l g w 9. The doubly charged molecular Ion of gramlcidln S (1 140 u) In the linear (A) and reflecting (B) modes.
RESULTS AND DISCUSSION Resolution in the Linear and Reflecting Modes. The first experiments in the linear mode were done in a 60-cm-long TOF instrument. They were repeatedlater in thespectrometer of Figure 1 with zero voltage on the mirror and ion flight times measured in the upper detector. The resolving power M /AMFWHM in the linear mode never exceeded a few hundred (300-500 depending on the ion mass and the ion source conditions). A direct comparison of the resolution in the linear and reflecting modes was carried out for gramicidin S (1 140.5 u) under constant ion source conditions. The time focusing for each mode was optimized independently. In the linear mode the best resolution was obtained when the ions were focused onto an object plane at the detector position; this required a small extraction pulse (-200 V). Theresolving power in this case was about 300 for the molecular ion peak (Figure 3A). When the extraction pulse was kept at 200 V, operation in the reflecting mode gave some improvement in resolution, but the isotopic distribution could still not be resolved. Thus conditions were far from optimum, as might be expected, considering the anomalous location of the object plane. The extraction pulse was then increased to 400 V to bring this plane close to the extraction region, as calculated above. Under this condition the optimum setting of the mirror voltage (4.5 kV) was found to be close to the full accelerating voltage, so the ions spent sufficient time in the mirror to obtain full velocity c ~ r r e c t i o n .As ~ ~ a result, the resolving power was greatly > 5000) and the isotopic peaks improved (to M/AMFWHM were clearly resolved, as shown in Figure 3B. Since the equivalent flight path has a length -3 m, the ion beam is compressed at the detector (and presumably also at the object plane) to -0.3 mm, compared to its -4-mm calculated width
Figure 4. The trlply char@ molecular Ions of (A) mellttin (2848 u) and (B) the B-chaln of bovine lnsulln (3498 u).
inside the storage gap. Examples of similar resolution in the reflecting mode at somewhat higher mass are shown in Figure 4.
-
The resolution was not changed significantly by slight variations (- 10 V) in the pulsevoltageor by variations 100 V in the mirror potential. However, for the remainder of the work reported here, voltages were kept fixed for the orthogonal acceleration (400-V pulse, 4-kV acceleration voltage) and for the mirror (4.5 kV). Adjustments were made only to the source parameters and to the deflection plate voltages. The improved resolution provided by the reflecting system is particularly useful in untangling a complicated spectrum. For example, Figure 5 (top) shows the spectrum of a tryptic digest of a recombinant protein IclR of mass -29 000 u, prepared by H. W. Duckworth and L. Donald of our Chemistry Department. Trypsin cleaves at arginine and lysine so a fragment normally contains only one of these basic residues, but in this case, the protein contained 13 histidines and the product masses ranged up to nearly 4000 u, so there was a reasonable probability of obtaining multiple charges on the fragments (up to 4+, as it turned out). Figure 5 (bottom) shows an expanded view of a small section of the spectrum, where the unit mass resolution enables the charge on each fragment to be determined. Thus the rather complicated spectrum of Figure 5 (top) is decomposed into four spectra corresponding to charge states I + to 4+, so interpretation is much easier. Variation of Resolution with Mass. Although in mass spectrometry resolution usually decreases with increasingmass, it might be expected to stay roughly constant for electrospray, since the charge tends to increase with mass so as to keep m/z in the same range of values. However, a noticeable deterioration of resolution has been observed in earlier ES/TOF instruments for masses greater than a few thousand u.’-I2
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1,304
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29,024 u
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FburoI. The mlzspecbumof a mdlgest of the IclR rscombhrent protein (28 932 u) (top) and the expandon of a small mlz regbn of thta spectrum (bottom).
Figure 4 shows two examples of molecular ion spectra of peptides in our spectrometer. For the peptides of masses up is about to =3500 u shown, the resolving power M/AMFWHM 5000,as measured for gramicidin S (Figure 3). As pointed out above, this resolution is sufficient to distinguish separate isotopic peaks for most products of the tryptic digestion of a large molecule and hence to determine their individual charge states. There may still be some structure visible on top of the peaks for molecules as large as bovine insulin (5733 u), but it is not clear enough to define the isotopic spacing, so the effective resolution is determined by the width of the isotopic distribution, i.e. M/AMFWHM-1000. For some larger proteins of masses up to -29 000 u, carbonic anhydrase for example (Figure 6),we have measured apparent resolving power from 1000to 1500. Theobservedvalues arelower limits because it is not known whether unresolved adducts are present or whether the proteins are completely homogeneous. Accuracy of Maas Determination. Figure 6 shows the deconvoluted spectrum of carbonic anhydrase 11. Its molecular weight was measured to be 29 024 u, using cytochrome c to calibrate the mass scale. This differs from the calculated mass by 2.5 u or 100 ppm, typical of the value achieved in our measurements for proteins of masses of 10 000-30 OOO u and similar to the accuracy obtained for the same compounds in our laboratory with matrix-assisted laser desorption. The factors limiting our accuracy in ES measurements are not yet understood, but we are investigating various curve-fitting programs to see if it can be improved.
Figuro 6. Carbonic anhydrase 11: mlz spectrum (A) and the deconvoluted mass spectrum (B).
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I
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1110 AnaWcaI Chemlsby, Vd. 66, Ab. 1, January 1, 1984
MlZ Figure 7. mlz speictfa for a 10" M solution of cytochrome c(12 361 u) at a flow rate of 0.01 &/e, consuming (A) 50 fmol In 5 s of remrdhg thne and (5) 10 fmd In 1 s of recording thne.
Sensitivity and Dynamic Range. Figure 7 shows two spectra of cytochrome c (12 361 u) recorded for the same water/ methanol solution at a protein concentration of 1 pmol/pL. The upper one was recorded for 5 s and the lower one for 1
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Flgm 8. mlr ap&alqof a modifiedangiotensln (883 u) accumulated molecular ion Is shown on a normalscab le expanded by a factor of 100 (8).
s, during which the amounts of protein consumed were 50 and
10 fmol, respectively. In spite of high losses of intensity at the slits and poor focusing after the skimmer, the instrument sensitivity is somewhat better than for most quadrupole instruments. This is a result of the high-duty cycle of pulse formation (-20%); nearly complete transmission of the pulsed beam through the TOF spectrometer, and simultaneous detection of all ions, i.e. no scanning. The dynamic range of the instrument is closely related to the sensitivity. Figure 8A shows the doubly charged molecular ion of a modified angiotensin (883 u), where the spectrum was recorded for 1 min and 3 pmol was consumed. The first four isotopes are visible. When the vertical scale is expanded by a factor of 100, as shown in Figure 8B, the fifth and sixth isotopic peaks appear, where the latter has an intensity 3 orders of magnitude lower than the first isotopic peak; i.e. it corresponds to 3 fmol. This large dynamic range is a benefit of the single ion counting mode of the recording system and of the purity of the ES spectra, free from decay fragments. Fragmentation. A useful feature of ES sources is the ability to produce controllable breakup of clusters and fragmentation of the ions in the region between the nozzle and skimmer by increasing the potential difference between these components and thus increasing the collision energy.%’ By examining the daughter ions produced in the collisions, considerablestructural information about the parent can be obtained. Figure 9 shows spectra of the peptide melittin (2846 u) containing 27 amino acid residues. Mild ionizationconditions produce only multiply charged molecular ions (Figure 9A), while higher collision energy yields extensive fragmentation (Figure 9B). The forest of peaks is rather forbidding at first sight, but our resolution is again good enough to determine the charge state of each daughter, producing considerable simplification. Assignments are noted in the figure, partic(27) FoIUman, J.; Reopstorff, P. Biomed. Mars Spectrom. 1984. 11, 601.
MIZ
Flgurr 8. mlz spectra of melittln (2846 u): unda mlld conditions (A) with nozzle to skimmer voltage Vw = 100 V and (By with VNs= 300 V, showing extensive fragmentatlon.
ularly the extensive series of singly charged B ions and doubly charged Y ions.27 The observed fragmentation is entirely a result of breakup taking place in the ES source between the nozzle and skimmer. No evidence of metastable decay in the first leg of the timeof-flight path has been observed for melittin, insulin,or smaller peptides. Metastable decay in the flisht path would produce charged or neutral daughters with less energy than the parent ion. However, no neutral fragments wereobserved in detector 1 behind the electrostatic mirror (see Figure l), and measurements with reduced mirror voltage showed no evidence for charged daughters. The absenceof significant metastable decay in the spectrometer is presumably a result of the time (-100 ps) spent in the source before ejection into the spectrometer, as found previously under analogous conditions by Cotter et al.I9 The spectra are therefore much cleaner than those observed in most TOF measurements, where ions enter the spectrometer immediately after being desorbed and excited. Soft Ionization. As seen above, harsh conditions in the ES source give considerable fragmentation, which can be useful in structural determination. Conditions at the opposite extreme (Le. as gentle as possible) give results that are even more interesting. Electrospray is the only technique that is mild enough to produce ions from weakly bound complexes, as shown in a number of recent investigations.28 However, it is not clear that every ES source/spectrometer combination is capable of producing such results, particularly when the ions must be accelerated to keV energies, as in our case. Nevertheless, we have been able to obtain the spectrum of myoglobin with its noncovalently bound heme attached, as illustrated in Figure 10. Similar results have been obtained for the a-chain of hemoglobin (16 742 u, including heme) and for carbonic anhydrase complete with Zn cofactor (29 087 (28) Sa,for example: Loo,J. A.; Giordani, A. B.; Muenster, H. Rapid Commun. Moss Spectrom. 1993, 7, 186 and references therein.
Analytical Chemlsby, Vol. 66,Ab. 1, &m&?ry 1, 1994
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150
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MIZ Flpuro 10. The molecular ion of myoglobin with Its heme retalned (17 587 u).
2000
6,000
3600
MI2 Flgure 12. Upper part of the population of multiply charged Ions from &galactosidase (113 600 u) In water/methanol (50/50) at pH -4.
50+
2Ooo
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3000
MI2 Flgure 13. m/zspectrumof mwine anti4buman al-ecklg l y ~ o t e i n ) monodonai antibody with a measved avermolecular of 149 800 f 100 u. The concentration was 3 X le7M in water wlth 10% acetic acM, and 3 pmol of protein was consumed WMO recordihg the spectrum.
E
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lo00
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MIZ F@wo11. mlr spectra of bovine albumln In water/methanol soiutlon (50/50) at lo4 M and (A) pH 2.5 and (B) pH -5.
u). Acceleration to keV energies is therefore no obstacle to the study of weakly bound systems, as found also by Loo et aL2* Thus our ES/TOF instrument appears to be well suited to investigation of these interesting entities. Mass/Charge ( m / z )Range. In principle the m / z range of a TOF spectrometeris unlimited; the only restrictions arise in ion production and detection. As remarked in the Introduction,the ions produced by ES sources usually lie within the m / z range of most quadrupole spectrometers (52400). However, an increasing number of exceptions to this rule are appearing. An example of ions with unusually high m / z is given in Figure 1 1, where the m / z distribution of the globular protein albumin (-66 000 u) is maximum at a value 1600 when sprayed from a solution of pH 2.5. However when sprayed under milder conditions (pH - 5 ) the maximum moves to 2200and peaks extend to m / z -4000. Additional examples are found in the m / z spectra of proteinase K and porcine pepsin, but a particularly interesting case is observed for @-galactosidase(- 114 000 u). For that compound, workers
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192 AmWml Ch8mistty, Vol. 66, No. 1, Jenuery 1, 1994
have reported m / z distributionswith a maximum at 1600.29 However, we find for similar solvent conditions that the m / z distribution is in fact bimodal.30 Figure 12 illustrates the upper part of the spectrum, which has a maximum at about m / z 3800 and extends almost to m / z 6000. In this high m / z range it is easier to resolve adjacent peaks. As a final example, h e show in Figure 13 the m / z spectrum of a monoclonal antibody of molecular weight close to 150 000 u. Feng and Konishi have examined this compound with a quadrupole spectrometer of m / z range 2400 but were only able to see the few highest charge states (above 63+) because of that Our spectrum has a maximum at about m / z 3000, and it appears that only a small fraction of the distribution lies below m / z 2400. The most abundant charge state is 50+. Thus we find that our ES source can produce ions with m / z values considerably above the quadrupole range and that these, as expected, can be examined in the TOF spectrometer. The way is therefore clear to study many interesting biomolecules and biomolecular complexes that possess much smaller charges than unfolded polar proteins. CONCLUSIONS In ES/TOF with orthogonal ion injection, the reflecting mode provides a significant improvement in resolution. (29) Jardine, I.; Hail, M.;Loris, S.; Zhou, J.; Schwartz, J.; Whitehowe, C. Proceedingsof the 38th ASMS Conference on Mass Spectrometry and A u i Topics, 1990; pp 16-17. (30) See,for a general discussion: Mirza, U. A.; Cohen, S. L.; Chait, B. T. Anal. Chem. 1993, 65, 1. (31) Feng, R.; Konishi, Y. Anal. Chem. 1992, 64. 2090; 1993, 65, 64s.
Although focusingand beam transport are not yet optimized in our source, the instrument attains a femtomole level of sensitivity. Unimolecular decay processes are almost entirely completed in the ES source before the ions are injected into the TOF spectrometer, so the observed spectra are very clean, even in the case where there is appreciable excitation of the ions. The ES/TOF instrument can be operated under conditions such that weakly bound complexes are not dissociated. Consequently it is well suited to examining these interesting entities. The large m/zrange of the ES/TOF spectrometer is likely to be useful in the study of biomolecules in relatively low
charge states. It appears that many of these can be produced in the ES source.
ACKNOWLEDGMENT This work was supported by grants from the National Institutes of Health (GMd060S) and from the Natural Sciencesand Engineering Research Council of Canada, which also providedm InternationalFellowshipto A.N.V. We thank Prof. A. Dodonov, Dr. I. Chernushevich, and Prof. B. Chait for fruitful discussions. Mr. V. Spicer and the University of Manitoba machine shop provided valuable technical support. Received for revlew June 24, 1993. 1993.@
Accepted October 20,
*Abstract published in Aduuncr ACS Absrrucrs. November 15, 1993.
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