Anal. Chem. 1990, 62, 1284-1295
1284
peaks in this series are of lower intensity and are observed up to m l z in 850. Two other sets of peaks observed in Figure 8b cannot be rationalized as arising from the ions produced by simple bond cleavages of Fomblin Z. These peaks have a spacing of m / z = 166, and their mlz values coincide with the two most intense Krytox series D-2 - [FRJ and N = [OR,CF,CFJ. They are attributed to Krytox impurities in the sample and are labeled D and N in the spectrum of Figure ab.
SUMMARY In summary, the TOF-SIMS spectra were obtained from thin films of Krytox and Fomblins Y and Z up to mlz = 6500. The fragmentation patterns were rationalized on the basis of the known structures and stabilities of the observed ions. Cationized Krytox oligomers were observed along with Krytox fragments. The fragmentation in the spectra of copolymers Fomblin Y and Z is complicated, and often several ion structures can be assigned to a given peak. The fragmentation patterns are unique for each kind of PFPE and are reproducible, independent of sample preparation and substrate. Several important points are demonstrated by this study. First, fragment ions of m / z > 300 in the perfluoropolyethers have intrinsic charge without cationization, unlike most other polymers. Second, quality spectra can be obtained from films ca. 1000 A thick, but these spectra contain peaks only due to fragment ions. Cationized oligomer ions occur only from films in the range of monolayer coverage. This demonstrates the importance of cationization for obtaining spectra from thick polymer films. Third, the study demonstrates the utility of TOF-SIMS for the structural characterization and the oligomer analysis of thin films of low molecular weight perfluorinated poly- and copolyethers.
ACKNOWLEDGMENT We thank J. D. Patton of E. I. du Pont de Nemours & Co. for providing the Krytox samples and for useful discussions. Registry No. 1, 25038-02-2; 2, 62168-88-1; 3, 64772-82-3.
LITERATURE CITED Krytox 143, 1500 and 1600 Series Fluorinated Oils. Technical Bulletin; DU Pont: Wlimington. DE. Fomblin Fluid Series. Technical Bulletin; Montedison Co.: MHano, Italy. Owno, A. C.; Appek, B. Org. Coat. Appl. Polym. Sci. R o c . 1981, 4 6 , 230. Cantow, M. J. R.; Larrabee, R. 6.; Barrail, E. M.; Butner, R. S.;Cotts, P.; Levy, F.; Ting, T. Y. Mkromol. Chem. 1988, 187, 2475. Brown, R. S.; Weil. D. A.; Wilkins, C. L. Macromolecules 1986, 19, 1255. Coburn, J. W.; Winters, H. F. J. Appl. fhys. 1986, 59, 1255. Au-Yeung, V. IEEE Trans. M g n . 1983, 19, 1662. Linder, R. E.; Mee, P. B. IEEE Trans. Magn. 1982, 18, 1073. Spikes, H. A.; Cann, P.; Caporiccio, G. J. Synth. Lubr. 1984, I , 73. Sinesi, D.; Zamboni, V.; Fontanelli, R.; Binaghi, M. Wear 1971, 18, 05. Baker. M. A.; Holland, L.; Laurenson, L. Vacuum 1971, 21, 479. Holland, L.;Laurenson, L.; Baker, P. N.; Davis, H. J. Nature 1972, 238,
--.
26
Holland, L.; Laurenson, L.; Hurley, R . E.; Williams, K. Nucl. Instrum. Methods 1973, 1 1 1 , 555. Pacansky, J.; Wakman, R. J.; Maier, M. J . fhys. Chem. 1987, 91, 1225. Bktsos, I.V.; Hercules, D. M.; Greifendorf. D.; Benninghoven, A. Anal. Chem. 1985, 57, 2304. Bletsos, I. V.; Hercules, D. M.; vanleyen, D.; Benninghoven, A. Macromolecules 1987, 20. 407. Steffens, P.; Niehuis, E.; Friese, T.; Greifendorf, D.; Benninghoven, A. J. Vac. Sci. Technol., A 1985, 3 , 1322. Bletsos, I.V.; Hercules, D. M.; vanleyen, D.; Hagenhoff, 6.; Neihuis E.; Benninghoven, A. Anel. Chem., submitted for publication. Bletsos, I. V.; Hercules, D. M.; vanleyen, D.; Benninghoven, A,; Karakatsanis, C. G.; Rieck. J. N. Anal. Chem. 1989, 61, 2142. Bletsos, I. V.; Hercules, D. M.; Magill, J. H.; vanleyen, D.; Niehuls, E.; Benninghoven, A. Anal. Chem. 1988, 60, 938.
RECEIVED for review November 6,1989. Accepted March 12, 1990. This work was supported by the National Science Foundation under Grant CHE-8541141, by the Bundesministerium fur Forschung und Technologie, by the Deutsche Forschungsgemeinschaft, and by a grant from the International Business Machines Corporation. We also acknowledge a grant from the Division of International Programs NSF, INT-8610485. We are grateful to the Alexander von Humboldt Foundation for a senior fellowship for D.M.H., which stimulated this work.
Electrospray Ionization Combined with Ion Trap Mass Spectrometry Gary J. Van Berkel, Gary L. Glish, and Scott A. McLuckey* Analytical Chemistry Diuision, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6365
Ions from a variety of molecules, formed vla electrospray, have been inbcted Into and analyzed wlth a quadrupole Ion trap mtrometer* are shown In which One Or more Of mass spectrometry (e*e'9mass opectrom etry'mass spectrom8try) have been pertormed On both mun@lychar*d and cations. for which data are Includethe disodium Of ncrphthalene-396-d'sUnon1c acid, MreCt Red 619 bradYklnln9 melmh CflOChme C 9 mY@Obfn, and b o v h albumin. For some COmPOdS, notably the wnmates, evidence 1s Presented for the Injection of highly solvated Ions that desolvate within the Ion trap. The cations derlved from the peptldes, on the other hand, appear to be essentlaly desolvated prior to Injection Into the ion trap.
INTRODUCTION One of the most significant advances in the field of mass spectrometry in recent years has been the development of various spray techniques for forming ions from analytes in solution. These techniques include, for example, thermospray (1-4) and electrospray (ES) (5-16). A remarkable feature of these techniques, particularly those that employ electric fields to impart charge to droplets in the spray, such as electrospray, is their ability to form highly charged ions from high molecular weight species (e.g., on the order of 100 protons have been observed to be attached to a molecule with a molecular weight of roughly 132000 ( 1 7 ) ) . These highly charged ions are characterized by masslcharge ratios that are well within the range of values accessible to most modern mass spectrometers.
0003-2700/90/0362-1284$02.50/063 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 13, JULY 1, 1990
The bulk of the mass spectra to date arising from electrospray have been acquired with quadrupole mass filters, although recent reports have described preliminary results from the coupling of an ES ionization source with a sector mass spectrometer (18) and with a Fourier transform mass spectrometer (19). Mass spectrometry/mass spectrometry (MS/MS) has also been applied recently to ions formed by electrospray (20-24) using triple-quadrupole mass spectrometers. Another important advance in mass spectrometry over the last few years has been the development of the three-dimensional quadrupole as an analytical mass spectrometer (25). This development was catalyzed by the introduction of the mass-selective instability mode of operation (26,27),the use of about 1mTorr of helium as a bath gas to improve sensitivity and resolution (28),and the commercial introduction of instruments designed to operate with these, and other, new features. The most sophisticated version of the commercial ion trap, the ion trap mass spectrometer (ITMS), is currently undergoing rapid development both in its capabilities as an analyzer and in its combination with other mass spectrometric techniques. Examples of the former developments include, for example, developments in methods for mass spectrometry/mass spectrometry (291, multiple stages of mass spectrometry (301,mass range extension (31-34), axial modulation (351,and automated gain control (36). Examples of the latter developments include, for example, methods for injecting ions formed in external ion sources (37-40), the coupling of laser desorption with the ITMS (41,42),and injecting ions from other types of mass spectrometers into ion traps (43-45). A number of features of the ITMS make it particularly attractive as the mass analyzer for ions formed by electrospray, particularly multiply charged ions. These include, but are certainly not limited to,high MS/MS efficiency, the capability for multiple stages of mass spectrometry (i.e., MS" where n > 2), and the capability for kinetic measurements. We have recently interfaced an ES ionization source with an ion trap mass spectrometer and report here our initial results, which are highly encouraging. Emphasis is placed on the injection, mass analysis, and detection of ions formed via electrospray. However, some MS/MS and MS" experiments have been performed on a preliminary basis and are briefly discussed.
EXPERIMENTAL SECTION Apparatus. The experiments were performed with a slightly modified version of an ITMS that has been adapted for the injection of ions from an atmospheric sampling glow discharge ionization source (46). This instrument and the results from ion injection experiments involving ions generated by glow discharge have been described (38). Figure 1shows a side-view schematic of the instrument used here (not drawn to scale). Solutions containing the analyte were pumped through a short length (about 50 cm) of 500-pm4.d. Teflon tubing at a rate of 1-10 pL/min with a syringe pump (Harvard Apparatus, Inc., Cambridge, MA) and then through a dome-tipped 120-pm4.d. stainless steel needle. The outlet side of the needle was placed 0.5-1.0 cm from the inlet aperture (marked A1 on Figure 1)of the interface, and a positive or negative potential, depending upon the ion polarity of interest, of 3-4 kV was applied to the needle. All elements in the interface region, viz., the plate containing the inlet aperture (Al) and the plate lenses housed between the aperture plates (AL1 and AL2, both having aperture diameters of 1.27 cm), were held at the same polarity as the needle. Aperture plate Al, which contains a 100-pm-diameter sampling orifice, was typically held at 150-200 V (same polarity as applied to the needle), and lenses AL1 and AL2 were adjusted for optimum ion signal. Optimum conditions were typically obtained with AL1 and AL2 both held within 150-200 V. Aperture plate A2, which contains an 800-pm-diameter exit orifice, was held at ground potential for all experiments. The interface region was held throughout at a pressure of 0.3 Torr. Although the "interface" was designed for glow discharge ioni-
5yr r a r
Pump
* ._
1285
C 0 i rnTorr (no He1
0 3 Torr n i
1
8
RlnQ Electrode A2
L2 S.S. k e d l e (120 I.Lrn i,d,)
Teflon Transfer Line
(500 w n 1.d.)
Detector
TT
&
Lenses
Endcap
8 LIsec
Endcap
350 T I s e c
Figure 1. Cross-sectional view of the instrumental configuration for
electrospray ionization at atmospheric pressure and ion injection into
the quadrupole ion trap. Drawing is not to scale.
zation, no experiments reported here involved the use of a glow discharge. The ion injection part of the experiment has been described (38). Briefly, during the ion injection portion of the experiment, ions issuing from the exit orifice of A2 are focused through the aperture in the ion entrance end cap of the ion trap via a three-element lens system (Ll, L2, and L3) situated between A2 and the end cap. At all other times, the ions that issue from A2 are deflected away from their path to the inlet aperture of the end cap via the application of a suitable voltage to one of the half-plates that constitute L2. Helium, which greatly facilitates ion injection and trapping in the ITMS, was admitted to the system to maintain a pressure of 1-3 mTorr for all studies reported here. Scan Parameters. A variety of experiments can be performed with the ITMS that involve various combinations of radio frequency (rf) and direct current (dc) voltages applied to the ion trap electrodes (47). The duration of application of each voltage and the overall sequence is referred to as the scan function. The most important signal for ion trap operation is the 1.1-MHz rf voltage applied to the ring electrode. It is this voltage that creates the trapping field within the ion trap, and its amplitude determines the mass/charge range of trapped ions (47). In the mass-selective instability mode of operation, the mass spectrum is acquired by scanning the amplitude of the rf voltage applied to the ring electrode and detecting ions as they successively acquire a value of the Mathieu parameter q of 0.908 (47). The q value for any given ion is linearly related to the ring electrode rf voltage amplitude. A t q values 2 0.908, ions exit the ion trap through holes in the centers of the end caps. The ions that exit through one of these end caps are then physically detected with a conversion dynode/electron multiplier detector. The application of a distinct rf voltage to the end caps of the ion trap during the experimental sequence has also been demonstrated both prior to (29) and during (32-35) the scanning of the ring electrode rf. For the experiments performed here, sine waves of equal amplitude and frequency, but 180° out-of-phase, were applied to the end caps. Frequencies were chosen to match what is commonly referred to as the fundamental secular frequency in the z-dimension, f,, which, under the conditions used in these studies, is closely approximated by f, = (21/2eZV)/(4~2mr02Q) (1) where Z is the integer number of charges, e is the unit charge C), V is the 0-p (p = peak) amplitude of the rf (1.602 X voltage applied to the ring electrode, m is the mass of the ion (in kilograms), r, is the inscribed radius of the ring electrode (1.0 x m), and Q is the frequency of the rf applied to the ring electrode (1.1 x lo6 Hz). Application of a sine wave with an amplitude of 0.1-2 V p-p and with a frequency off, for an ion of a particular mass/charge before the scan of the ring electrode rf amplitude has been shown
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 13, JULY 1, 1990
Table I. Summary of Peptide Data compd
av MW
species detected
leucine enkephalin bradykinin
555.6 1060.2 2 846.5 5 732.4 12 360 16 950 66 000
(M + H)+ (M + 2H)2+ (M + 4H)*+ (M + 5HY+ (M + nH)"+,t~ = 12-17 (M + nH)"+,n = 14-25 (M + nH)"+,n = 32-51
melittin
bovine insulin cytochrome c myoglobin bovine albumin
flow rate, pL/min
sample concn, PmollrL
figure of merit," fmol
0.5
65 18.9 7.7 7.7 3.6 4.8 2.4
1.1 0.6
1.0 10.0 1.0 1.0
1.5 1.0
32 1.3 1.2 10
1.0
@Thefigure of merit represents the quantity of analyte that passed out of the capillary needle during the minimum ion injection period of a single scan of the ion trap necessary to give analyte ion signals 5 times in excess of the standard deviation of the noise. The figure of merit may or may not represent the detection limit for the analyte depending upon the overlap in time of sample ionization and ion injection. to be effective in increasing the kinetic energy of the ions with that mass/charge value. This increase in kinetic energy, coupled with inelastic collisions with the helium bath gas, often results in the collision-induced dissociation (CID) of the ions (29). This form of ion activation is the most frequently used method for causing ions to fragment between stages of mass analysis in MS/MS and MSn experiments in the ITMS. Application of a sine wave with an amplitude of at least 6 V p-p to the end caps during the scan of the ring electrode rf amplitude is referred to as "axial modulation". This is effective in causing the ions to exit the ion trap before reaching the q value of 0.908. When the frequency of the supplementary voltage is chosen to excite ions just slightly greater in masslcharge than the low mass cutoff, in effect at the initiation of the ramp of the ring electrode rf amplitude, ions still exit at near q = 0.908. This procedure has been shown to improve mass resolution in normal ion trap operation (35).When the supplementary rf frequency is chosen to excite an ion significantly greater in mass/charge than the initial low mass cutoff, ions can be forced to exit at much lower values of q (and, hence, at lower ring electrode rf amplitudes). This procedure has been shown to be effective at extending the mass/ charge range of the ITMS from its normal value of 650 to as much as 30000 (32-34).For the studies discussed here, the mass/charge range has only been extended by a factor within the range of 1-10 (Le., mass/charge 650-6500). A dc voltage is sometimes added to the rf voltage applied to the ring electrode (47). This is usually done to isolate ions of a particular mass/charge value in MS/MS and MS" experiments. All of the voltages described above have been employed at some point in these studies. For mass spectra, only ring electrode rf and axial modulation were employed. Those cases are indicated in which data were acquired with significant extension of the mass range by axial modulation. Appropriate dc voltages were used during the scan function to effect ion isolation in MS/MS and MS" experiments. Between stages of MS, a supplementary rf signal was applied to the end caps to exite ions of the mass/charge of interest. Unless otherwise noted, values for the duration and amplitude of the supplementary rf used to induce fragmentation were 5-30 ms and 0.2-2 V p-p, respectively. Samples. All analytes were obtained commercially and were used as supplied. Tetrabutylammonium iodide, the disodium salt of 2-hydroxynaphthalene-3,6-disulfonicacid, and Direct Red 81 were all dissolved in HPLC-grade methanol. The respective solution concentrations are indicated in the relevant parts of the text. All of the peptides were dissolved in solvent mixtures comprised of HPLC-grade water, methanol, and glacial acetic acid in relative approximate proportions of 20%, 75%, and 5% by volume, respectively. Solution concentrations and flow rates used for each peptide are listed in Table I.
RESULTS AND DISCUSSION The results obtained for ES with the ITMS using the present interface are illustrated first with negative ion data including both mass spectra and MS/MS and MS" results. These results are followed with a presentation of similar types of data obtained for positive ions with particular emphasis on mass spectral results for relatively high molecular weight species. The results presented here were selected to indicate
the current level of performance of this combination and prospects for future applications and to illustrate the phenomena that have thus far been observed for the injection of ions formed by ES into an ITMS. Several aspects of this work are common to experiments with both ion polarities. For example, a noteworthy observation for mass spectra is the relative lack of cluster ions either containing the analyte species or comprised of solvent molecules only. In every case studied to date, the base peak in the ES/ITMS mass spectrum is due to an unsolvated analyte ion that is expected to be present in solution. Certainly part of the reason for the relative lack of solvent-only-containing ions is that the optimum conditions for injecting ions of high mass/charge discriminate against the injection and trapping of low mass/charge ions (38). Instrumental mass/charge discrimination effects should not, on the other hand, play a major role in the observation, or lack thereof, of analytecontaining cluster ions. In a few cases, we have observed adduct ions that contain the analyte with one water molecule or one methanol molecule, and in rare instances, ions of low abundance have appeared that contain the analyte with more than one solvent molecule. This is, perhaps, noteworthy because neither a heated bath gas, which some workers have used to minimize clustering in ES (6),nor a nebulizer gas, which some workers use to assist in desolvation (25), was employed with the ES interface in this work. The fact that relatively few cluster ions that contain the and@ are observed with the instrument described here can be attributed to a combination of several factors, which are expected to come into play to varying degrees depending upon the analyte and the instrumental conditions. First, the interaperture region of the interface is not collision-free. The Mach disk associated with the expansion from the 100-pm inlet orifice into the intermediate region maintained a t 0.3 Torr is 0.34 cm, while the interaperture distance is 1.9 cm. Under these conditions, an ion can undergo multiple collisions (greater than 10) with the background gas molecules in the intermediate region before it reaches the exit orifice. Therefore, the cooling and possible clustering that occurs upon the initial expansion is counteracted by the subsequent collisions in the intermediate region. (A reviewer has pointed out that scattering in the interface region may lead to discrimination against low-mass ions (e.g., solvent-only ions).) Second, clustering in the expansion from the 800-pm exit orifice into the vacuum system is much less likely than it is for the expansion a t the inlet orifice due to the much lower number densities in the former. Third, when the analyzer region is held at a helium pressure of several millitorrs, collisions with helium can occur prior to reaching the entrance end-cap electrode of the ion trap. Fourth, fragmentation associated with the injection of ions into a quadrupole ion trap has been noted for some ions (37-40). Evidence has been presented indicating that fragmentation can be induced either by collision with the back-
ANALYTICAL CHEMISTRY, VOL. 62,NO. 13,JULY 1, 1990
ground helium or by collision with an end-cap surface (44). It therefore seems reasonable to expect that, in some cases, the collision phenomena that underlie fragmentation upon injection can play a role in the removal of solvent molecules from an ion injected into an ion trap. Finally, the conditions under which ions are trapped in the ITMS may be conducive for desolvation. Extensively solvated ions injected into the ion trap may shed solvent molecules prior to mass analysis. This process may be assisted by collisions with helium. It is known that the kinetic temperatures of trapped ions can be well above room temperature (48-50). Under the multiplecollision conditions normally used in the ITMS, highly solvated ions may be "heated" internally while they are being kinetically "cooled" by collisions with helium, thereby accelerating desolvation. In any event, strong evidence that desolvation occurs within the ion trap for some of the ions discussed here is presented below. System performance, with regard to the quantity of analyte required for analysis, is determined by calculating the quantity of sample necessary to give a signal at least 5 times that of the standard deviation of the background noise. In those cases in which only a single scan is taken to obtain a spectrum (Le., no averaging), the quantity of sample consumed is taken to be the calculated amount of analyte that flows from the capillary needle during the ion injection period of the experiment, i.e., during the period of time that ions are allowed to accumulate in the ion trap. This is simply the product of the analyte concentration, flow rate, and injection time. It is assumed that equivalent mass spectra could be obtained from equal quantities of analyte whether the analyte flows through the capillary at a constant concentration during the injection period or the analyte passes through the needle in a peak, as long as the peak exits the needle during the ion injection period. I t should be realized that this quantity is not necessarily the minimum amount of material that must be subjected to mass spectrometric analysis. It only applies to those cases in which the passage of the analyte out of the capillary matches or falls within the ion injection period. Any ions formed outside of the ion injection period, such as during the scan of the ring electrode rf amplitude, are not measured. Therefore, in those cases reported here in which several scans are averaged to give a single spectrum, the quantity of sample consumed is determined by using the total time to acquire all of the spectra. The system is typically optimized for injection of ions formed via ES by spraying a solution of tetrabutylammonium iodide in methanol (roughly 5 X M) at a flow rate of 1-5 pL/min. Operating conditions for negative ions are optimized by monitoring the I- signal while those for positive ions are optimized by monitoring the tetrabutylammonium cation signal. Under optimized conditions, the I- signal from an 18 ng/pL methanol solution flowing through the capillary a t a rate of 2.5 pL/min can be detected with an injection time of 10 ms. The quantitiy of tetrabutylammonium iodide that flows from the capillary needle during this period is 20 fmol. The tetrabutylammonium cation can be detected from the same solution, under optimized positive ion conditions, at levels slightly under 10 fmol. Negative Ion Mass Spectra. Mass spectra of anions formed via electrospray were acquired for several relatively small compounds expected to yield anions in solution. An example is given in Figure 2, which shows the negative ion ES mass spectrum of Direct Red 81, a sulfonated azo dye (structure shown below in Scheme I), which has been shown to form (M - Na)- and (M - 2Na)" anions, among others, via thermospray ionization (51). The normal mass range of the ITMS was extended by a factor of 6 by using axial modulation to acquire this spectrum. The base peak in the spectrum is
1287
Ili, 350
450
550
650
mlz Flgure 2. Negative ion ES mass spectrum of Direct Red 81. Approximately 2 pmol of analyie flowed from the capillary needle during the data acquisition period.
(M-2Na)*'
151
I
ll
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 13, JULY 1, 1990
.
a31
. U
(a) no delay
I : 0
I.-.*. * . . . ' . . 0
' . . . ' . . .' . ' . ' . . . ' 100 200 300 400 500 600 700 '
Low rn/z Cutoff Duing ion Injection
Flgure 4. Plot of the relative abundance of (M - 2Na)'- from the disodium salt of 2hydroxynaphthalene-3,6disulfonic acid ( m / z 15 1) as a function of the low mass cutoff during Injection. In all cases, the rf amplitude was dropped to a low mass cutoff of m l z 60 following injection and held there for 100 ms to facilitate ion desolvation.
However, evidence presented below indicates that the dianion is not initially injected into the ion trap as a "dry" ion but rather as a highly solvated anion that undergoes extensive desolvation within the ion trap. This interesting phenomenon has been observed for several, but not all, compounds that we have analyzed. The disodium salt of 2-hydroxynaphthalene-3,6-disulfonicacid is used here as an illustrative example. Perhaps the most striking initial observation that suggested that highly solvated anions were being injected into the ion trap was the high rf amplitudes that could be applied to the ring electrode during the injection period. Our experience in injecting anions with m / z ratios within the range of 16-450 has indicated that the optimum rf amplitude during the injection period for an ion of m / z 151 should be about 320 V 0-p, which corresponds to a low mass/charge cutoff of 28 (Le., q = 0.908 for an ion of m / z 28) (38),and that the injection efficiency for this ion should be essentially 0 at rf amplitudes greater than 570 V 0-p (low masslcharge cutoff of 50). We find, however, that intense signals due to (M - 2Na)2- can be observed when rf amplitudes much higher than 570 V 0-p are used during injection. In fact, a broad maximum is observed for the optimum rf level during injection that falls well above the cutoff for m/z 151. Figure 4 shows a plot of the relative signal observed at m / z 151 as a function of the low mass/ charge cutoff, as determined by the ring electrode rf amplitude employed during the injection period. In all cases, the rf amplitude was dropped to a low mass/charge cutoff of 60 after ion injection and held for 100 ms prior to acquiring the mass spectrum (see below). The fact that the m / z 151 ions are detected even when the low masslcharge cutoff during injection is substantialy higher than m/z 151 shows conclusively that ions with masslcharge ratios greater than 151 are initially injected into the ion trap. Further evidence that desolvation occurs within the ion trap for these ions comes from a pronounced effect of trapping time on the intensity of the m/z 151 signal. This is demonstrated rather dramatically in Figure 5, which compares the mass spectra obtained by using a 2-ms injection time (at an rf amplitude corresponding to a low masslcharge cutoff of 200) and obtained with and without a 100-ms delay period before the analytical scan of the rf amplitude. In both cases, the rf amplitude is dropped to a low masslcharge cutoff of 60 immediately after the injection period and the mass spectrum is scanned from m / z 60 to m / z 400. The interpretation for these observations is that highly solvated ions with a range of masslcharge values are accumulated in the ion trap. If the mass spectrum is acquired before ions can desolvate, no ions fall within the mass range of the instrument and, therefore, no signals are observed. However, given sufficient time for
130
150
170
190
210
190
210
mh
83
(M-2Na)'' 151
1
130
150
170
m/z Flgure 5. Negative ion ES mass spectra of the disodium sal of 2hydroxynaphthalene3,6disuifonicacid obtained with (a) no delay perkd for desolvation after injection and with (b) a 100-msec delay after injection. The low mass cutoff during ion injection was m / z 200.
desolvation, the ions can completely "dry", yielding the dianion at m / z 151. Further support for this interpretation comes from an experiment that uses the mass-selective storage capability of the ion trap. When rf and dc voltages of the appropriate values to reject all ions with mass/charge values other than m / z 151 are applied to the ring electrode immediately after the ion injection pulse, essentially no ions are observed in the mass spectrum regardless of the delay time used after the rf/dc period. This result indicates that the high-mass ions that must be initially present in the ion trap are rejected by the combined rf and dc voltages. There are, therefore, no ions remaining in the ion trap to shed their solvent molecules to give the dry dianion. Desolvation within the ion trap is an interesting phenomenon that itself merits further study, and the ITMS is wellsuited to measuring the overall kinetics for this process. For example, Figure 6 shows the normalized dianion intensity observed as a function of the delay time after ion injection (i.e., desolvation time) by using the same scan and injection parameters employed for the spectra shown in Figure 5. Desolvation appears to be complete within 120 ms, as is also observed with dianions from Direct Red 81 sprayed from a methanol solution. We have noted, however, that several experimental variables appear to affect the time for complete desolvation. These include, for example, the ring electrode rf amplitude during the delay time and the pressure of the bath gas. It is presently unclear to what extent desolvation is accelerated by the presence of the bath gas. The data collected to date do not clearly separate the effects of the bath gas on injection efficiency and on desolvation. These effects, as well as the effect of the nature of the bath gas and the nature of the solvent system, will be subjects of further study.
5
0.60
c 1
0.40
,+
>.
=! i 4-
o.20 0.00
.
.
.
. .
ANALYTICAL CHEMISTRY, VOL. 62, NO. 13, JULY 1, 1990
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(M-2Nar '151
I
5071
2
0.03 0.05 0.07 0.09 0.1 1 0.13 0.15 Desolvation Time
(sed
K
Figure 8. Nom?aked abundance of (M - 2Nay- from the disodium salt of 2-hydroxynaphthalene-3,6disuifonic acid as a function of the delay time (desolvation time) after ion Injection. The scan function and injection parameters for this experiment are the same as those used to acquire the data in Figure 5.
Negative Ion MS/MS and MS" Studies. Once anions are injected into the ion trap and stored, the various means for manipulating ions with an ITMS are available. MS/MS and MS" experiments are clearly among the most useful that can be performed with the ITMS. Although MS/MS and MS" of multiply charged ions formed by ES is not the main thrust of this communication, we report here a few examples of MS/MS and MS" of some of the ions formed in these studies. The (M - 2Na)2- ions from Direct Red 81 and 2-hydroxynaphthalene-3,6-disulfonicacid serve as the parent ions to illustrate what we believe are the first MS" experiments performed on dianions in a quadrupole ion trap. Figure 7 shows the ES/MS spectrum of 2-hydroxynaphthalene-3,6-disulfonic acid following a mass selection step to isolate (M - 2NaI2-, the MS/MS spectrum obtained following kinetic excitation of (M - 2NaIz-, and the MS/MS/MS spectrum obtained after isolating and kinetically exciting (M - 2Na - SO3')'- formed in the preceding MS/MS experiment. CID of (M - 2Na)2- results predominantly in a charge separation fragmentation to give SO3'- and (M - 2Na - SO3'-)'(Figure 7b). CID of the latter ion results in the loss of SOz to give the anion at m / z 158, with the probable structure shown (Figure 7c). The major CID reaction observed for (M - 2Na)2- from Direct Red 81 (Figure 8) also involves charge separation, but unlike (M - 2Na)2-from 2-hydroxynaphthalene-3,6-disulfonic acid, the masses and charges of the primary daughter ions do not sum to that of (M - 2Na)2-. As shown in Figure 8b, the two major daughter ions in the MS/MS spectrum of (M 2Na)2- fall at m / z 260 and m / z 341, respectively. The fact that these masslcharge values are not equidistant from that of the parent ion at m/z 314.5 (Figure 8a) indicates either that the two ions are not formed from the same reaction. or that a neutral species is lost in conjunction with charge separation. The reasoning and experiments discussed below indicate that the latter situation is most likely. The daughter ion at m / z 341 is clearly singly charged since its masslcharge is greater than that of the parent ion. The conjugate singly charged daughter ion must, therefore, be of masslcharge less than that of the parent and should, if detection and trapping efficiencies are similar (and no other reactions occur), be observable at comparable abundance. The only other abundant daughter ions are observed at m/z 260 and at m/z 156. The ion at m/z 156 is a granddaughter ion produced from the m / z 260 daughter ion, as indicated in the MS/MS/MS spectrum (Figure €k)produced by subjecting the parent ion and the m/z 260 daughter ion to CID in sequence. The m / z 156 daughter ion in the MS/MS spectrum, therefore, probably arises from further fragmentation of the mlz 260 ion. The fact that the s u m of the signals at m/z 156 and m/z 260 is essentially equal to that of the m / z 341 fragment is consistent with this in-
100
150
200
250
mlz (M-2Na-S03-*)
Im
li3~'OL
(b) MS/MS 151
0
1
0
m/z
I
t
160
fc)
200
180
MS/MS/MS
220
mh Figure 7. Negative ion ES mass spectra of the disodium salt of 2hydroxynaphthalene-3,6disuifonic acld showing (a) the spectrum obtained by rf/dc isolation of (M - 2Na)'-, (b) the MS/MS spectrum of (M - 2Na)'-, and (c) the MSlMSlMS spectrum of (M - 2Na)'-.
terpretation. Scheme I shows a fragmentation scheme that would account for the data. A recent publication describing MS/MS data obtained for a variety of multiply charged anions, including some derived from azo dyes, has also concluded that the loss of Nz, as well as SO3'-, frequently occurs from azo dye anions (24). Positive Ion Mass Spectra. In collecting positive ion ES mass spectra with the ITMS, emphasis has been placed on the ionization of peptides due to both the widespread interest in these compounds and the known propensity for multiple protonation of these species during ES. Mass spectra for all the peptides reported in this study have been reported in the literature using ES combined with a quadrupole mass filter. Unlike the dianions discussed above, all of the peptide cations were optimally injected at relatively low rf amplitudes on the ring electrode, and ion signals were not enhanced by use of
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 13, JULY 1 , 1990
Scheme I
(M-2Nar 1
(M-2Na)"
'314.5
-0,s
mlz 314.5
I
mlz
(b)
MS/MS
341
MWMS 314.5 0
1 0
~
+ 2
N,
+
m/z 260
MSIMSIMS
mlz 341
156
150
200
250
300 mlz
350
400
(c) MS/MS/MS 314.5 0
1
260
0
I
0 156
260
150
200
250
300
mlz Figwe 6. Negative ion ES mass spectra of Direct Red 81 showing (a) the spectrum obtained by rf/dc isolation of (M - 2Na?-, (b)the MSlMS spectrum of (M - 2Na)*-, and (c) the MS/MS/MS spectrum of (M 2Na)l-.
a delay period after ion injection and prior to mass analysis. These observations indicate that the peptide cations are essentially desolvated prior to being trapped. Table I lists the peptides that have been ionized via electrospray and injected into the ITMS, the major ions observed, the flow rates and the concentrations employed, and what is referred to as a "figure of merit". The figure of merit represents the minimum quantity of material necessary for detection of the analyte in a single scan as determined by the product of the injection time, flow rate, and solution concentration. The quantities in the latter category may or may not represent the minimum quantity of material required for mass spectrometric analysis. Only under conditions where analyte introduction and ionization are completely enveloped
mlz 156
in time by the ion injection period of the experiment will the figure of merit represent the detection limit for the analyte. In analogy with pulsed ionization experiments, detection limits are optimized when sample introduction is synchronized with ionization. The determination of detection limits for these compounds under various sample introduction scenarios is not an objective of this report. Nevertheless, the measured figures of merit certainly indicate that the ES/ITMS combination will provide sufficiently low detection limits to be analytically useful. The remainder of the discussion of the positive ion mass spectral data is devoted to illustrating the behavior of the present ITMS system as an analyzer for the peptide ions. The nominal upper mass/charge limit of the ITMS, when operated in the usual way, is 650. However, the effective upper mass/charge limit of the ion trap used in this work was restricted to about 400 due to a limitation of the rf amplitude that could be applied to the ring electrode, which was imposed by the use of relatively dirty ceramics to isolate the end caps. For this reason, all positive ion mass spectra were acquired by using axial modulation to eject ions from the ion trap before they reached a q value of 0.908, as described in the Experimental Section and elsewhere (34). For example, Figure 9 shows the mass spectrum obtained from roughly 50 fmol of bradykinin (MW = 1060) by using axial modulation to eject ions from the ion trap at one-half of the ring electrode rf amplitude necessary to eject the ions without axial modulation (i.e., double the mass/charge range). Bradykinin forms a dication predominantly (i.e., (M + 2H)2+)that is observed at m / z 531. It was noted that for the higher molecular weight compounds (>lo*),which give a cluster of multiply charged ions, maximum signal intensities were observed by using mass scale multiplication factors that force the ions to exit the ion trap at ring electrode rf amplitudes that are ordinarily necessary to eject ions of mlz 80-200 without axial modulation. For
ANALYTICAL CHEMISTRY, VOL. 62, NO. 13, JULY 1, 1990
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U
supplementary r f amplitude
400
450
500
600
550
mlz Figuo Q. Posithre ion ES mess spectrum of bradykinin. Approximately 50 fmol of analye flowed from the capillary needle during the data
acquisition period.
I
,
1000
,
( ,
.
,
,
,
,
,
1800
1400
.
/
.
,
2200
m/z
Flgure 10. Positive ion ES mass spectrum of bovine albumin. Approximately 124 fmoi of analyte Rowed from the capHlary needle during the data acquisition period. this reason, the mass spectra obtained for the larger peptides were acquired by using mass range extension factors of 6-10. For example, Figure 10 shows the ES mass spectrum obtained for bovine albumin (MW = 66000), the highest molecular weight compound we have attempted to analyze. This mass spectrum was acquired by applying axial modulation at a frequency necessary to eject ions at 1/10 of the ring electrode rf amplitude normally required to remove these ions (mass/charge range 1300-2000) from the ion trap. It is noteworthy that high-mass, high-charge ions can be injected from the ion trap and subsequently detected even by forcing ions to exit the ion trap at ring electrode rf amplitudes as low as 1/10 of the value required in the absence of axial modulation. However, since the ITMS scans the rf amplitude at a fixed rate of 30 ps/data point, and ions are forced to exit the ion trap over a narrower rf amplitude range, this approach effectively increases the scan rate in terms of daltons/second, with potential adverse effects on resolution and peak height measurement (see below). Furthermore, a limitation with the present system stems from the fact that the use of mass range extension by axial modulation reduces the number of data pointa/dalton, thereby compromising the accuracy of the mass measurement. There are about 0.55 data points/V of rf amplitude in the present ITMS system, and without mass range extension, there are about 11.5 V/Da giving about 6 data points/Da. Mass range extension forces more daltons within the same rf voltage window, thereby reducing the number of data points/dalton by the mass range extension factor. A reduction in the number of data points/peak compromises the accuracy of the assignment of peak position. As shown below, this can have a significant effect on both the determination of molecular weight and the charge for high-mass, multiply charged ions.
Flgure 11. Dual rf scan function used to establish that ail ions are removed from the trap by using axial modulation during the data acquisition ramp of the rf amplitude. The same fixed frequency was applied to the end caps during both rf ramps. The scan speed was fixed for the second ramp but was varied for the first ramp. The mass resolution observed in these studies for the multiply charged, high-mass ions, defined as the mass of the ion divided by the peak width at one-half of the peak height in mass units (M/AM), was typically 500-600. This is close to the normal resolution afforded by the ITMS for singly charged ions. The use of axial modulation to increase the mass range of the ion trap involves bringing ions successively into resonance with the frequency f, applied to the end caps. The observed resolution is therefore determined by the frequency range over which an ion absorbs kinetic energy sufficient to exit the ion trap. Studies to determine the factors that affect mass resolution using this approach for high-mass, multiply charged ions will be performed when measures can be taken to increase the number of data points/peak and to control the scan speed. The use of axial modulation to eject ions from the ion trap, unlike the mass-selective instability method, relies on ions being ejected during the period in which they pass through a resonance condition. It is therefore possible to scan the ring electrode rf amplitude too quickly to allow all of the ions to attain the necessary kinetic energy to exit the ion trap. We have used the scan function shown in Figure 11to establish that all of the ions investigated in this study were completely removed from the ion trap by using this form of axial modulation. The scan function includes two scans of the ring electrode rf amplitude during which the same fixed frequency was applied to the end caps to extend the mass range. The data system does not allow the scan speed to be varied for the second rf ramp, but the slope of the first ramp can be increased by reducing the time of the ramp. If the scan speed is sufficiently slow to eject all of the ions of interest, the first ramp should remove all of the ions and, therefore, no ions would be detected during the second ramp. If, on the other hand, the scan speed is too fast, ions will be detected during the second ramp, since they are not removed by the first ramp. We have found that, in every case, the first ramp was 100% effective in removing the ions when its scan speed was equal to that of the second ramp. We are currently systematically studying the conditions under which ions are not effectively removed. Mass/charge measurements with an ion trap have been made by a number of methods (47). The most convenient approach in the present system involves scanning the ring electrode rf amplitude while applying a fEed rf frequency (and amplitude) to the end caps and measuring the ring rf amplitude necessary to bring ions into resonance with the rf applied to the end caps. The resonance condition is indicated by applying a sufficiently large rf amplitude to the end caps to cause the ion to be ejected from the ion trap and detecting ions that strike an external ion detector. The correlation between ring rf amplitude and mass/charge ratio for the ITMS, in the absence of axial modulation, is ordinarily determined by using the electron ionization mass spectrum of a perfluorinated compound such as perfluorokerosene. However, since no filament is present in the current ES/ITMS system, we have used the mass spectrum of cytochrome c (MW
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WhJVW' 700 ' 800
1000
900
11'00
m/z
Table 11. Summary of Myoglobin Mass Calculations
rnlz
708.4 f 737.1 f 770.7 f 810.6 f 851.2 f 893.9 f 942.9 f 998.2 f 1059.1 f 1131.9 f 1212.4 f
1.169 1.169 1.169 1.169 1.169 1.169 1.169 1.169 1.169 1.169 1.169
900
1100
mlz
Flgurr 12. Positive ion ES mass spectrum of cytochrome c . Approximately 12 pmol of analyte flowed from the capillary needle during the data acquisition period.
cor
700
calcd z
assigned z
24.65 f 1.42 21.29 f 1.08 19.29 f 0.80 19.94 f 0.81 19.91 f 0.77 18.22 f 0.62 17.03 f 0.51 16.37 f 0.44 14.53 f 0.33 14.05 f 0.29
24 23 22 21 20 19 18 17 16 15 14
calcd MW 16980 f 16950 f 16930 f 17000 f 17000 f 16970 f 16950 f 16950 f 16930 f 19960 f 16960 f
28 27 26 25 23 22 21 20 19 18 16
16960 f 30 av
= 12 360) to calibrate the mass/charge scale over the range of rf amplitude voltages that, in the absence of axial modulation, eject ions of m/r 100-180. This spectrum, shown in Figure 12, was obtained by using axial modulation to eject ions from the trap at one-sixth of the ring electrode rf amplitude necessary to eject ions in the absence of axial modulation. The charge calculated for each peak in the cytochrome c spectrum was determined by using the uncalibrated mass/charge scale following the method described by Covey et al. (16). The mass scale calibration obtained with cytochrome c was used to determine the mass/charge values of the peak maxima observed in the mass spectrum of horse skeletal muscle myoglobin (Figure 13). Table I1 shows the observed mass/charge values from the calibrated mass scale, the calculated charge, the assigned integer charge, and the molecular weight determined from each peak (obtained from the product of the observed mass/charge and integer charge minus the mass of the number of protons corresponding to the integer charge). The average molecular weight of myoglobin, based on the molecular formula, is 16 950.4. The molecular weight obtained by averaging the values obtained from the individual peaks is 16960 with a standard deviation of 30 (relative standard deviation of 0.15%). The precision of this measurement is limited by the fact that the mass/charge of the peak maximum, as it appears on the mass scale of the data system, can only be determined, at best, to within 1data point and there are only about 6 data points per mass/charge division. Therefore, the mass/charge measurement is limited to four significant figures with the final place uncertain by a t least 0.167 mass/charge units. The uncertainty in the molecular weight assignment from each measurement is, therefore, in the tens. (The peak maximum should actually reflect the mass of the most abundant isotope, rather than the average mass. The average mass would be more accurately determined from the peak centroid. However, the average mass exceeds that of the most abundant isotope only by about
Figure 13. Positive Ion ES mass spectrum of myoglobin. Approximately 213 fmd of analyte flowed from the capillary needle during the data acquisition period.
1 Da (MW = 16 949.5). Considering the error in assignment of peak position and the limited number of data points/peak, centroiding in this case is unjustified). The uncertainty in the mass/charge assignment due to the limited number of data points/peak also significantly affects the determination of the number of charges associated with the molecule. The formula to obtain the number of charges associated with an ion from the measured mass/charge values of two adjacent peaks, using axial modulation to extend the mass scale by a factor of 7, is
where n2is the charge associated with the higher mass/charge peak; m, and m2 are the values of the lower and higher mass/charge peaks (as they appear on the corrected mass scale from the data system), respectively; and X is the mass of the cationizing species. All of the cations associated with the molecules are assumed here to be protons. The standard deviations associated with the charge assignment for the ions in the myoglobin spectrum, assuming the mass/charge assignment can be made with a standard deviation of 0.167 mass/charge units (which corresponds to the interval between data points), are included in Table 11. The relative standard deviations for the charge assignments are much larger than those for the molecular weight assignments due to the difference taken in the denominator of eq 2. In a few cases, the calculated charge is inconsistent with the trend established by the ions of lower charge (where error in charge assignment is smallest). These.deviations could be due to either the experimental uncertainty in peak position measurement or the presence of cations other than protons in some of the ions. Multiple cationization with species such as sodium and potassium, under appropriate solution conditions, has been observed in ES (52). Mixtures of cations could account for some of the deviations of the experimentally derived molecular weights and numbers of charges from the actual values. However, given the mass resolution and the uncertainty in mass/charge assignment, we cannot determine if this situation exists for the high-mass, high-charge species. Furthermore, the experimentally derived values generally agree with the actual values within the uncertainties of our measurements, so that invoking the presence of cation mixtures in some of the ions is unjustifiable. Clearly, it is highly desirable to reduce the error in mass/charge measurement. Both the molecular weight assignment and the charge state assignment could then be made with greater confidence, and the presence of cation mixtures would be more readily discerned. The obvious way ko improve mass and charge assignments, made by measuring the rf amplitude a t which ions are ejected from the ion trap with the
ANALYTICAL CHEMISTRY, VOL. 62, NO. 13, JULY 1, 1990 1293
(M+4H)4+ 712.6 7
6431
I
B', IY.."
2+ + Y18 Jll 2+ + y19 1 4 2
500
'
700
'
900
I
11100 IO0
mlz
Figure 14. Positive ion ESIMSIMS spectrum of (M + 4H)" from melittin. Approximately 4 pmol of analyte flowed from the capillary needle during the data acquisition period.
present system, is simply to increase the number of data points/peak, and efforts are under way to do this. Other approaches to detect the resonance condition may prove to be superior to axial ion ejection for the high-mass, multiply charged ions. For example, early approaches to mass/charge measurement in quadrupole ion traps used power absorption from the rf circuit that supplied the end-cap rf voltage (53, 54),in analogy with some ion cyclotron resonance experiments (55). Syka and Fies (56) have recently used image current measurements in conjunction with Fourier transform techniques with an ion trap, in analogy with Fourier transform ion cyclotron resonance experiments. The measurement of secular frequencies, using relatively low rf power supplied to the end caps, might provide a superior approach to mass measurement for the high-mass, high-charge ions than the measurement of the ring rf amplitude made in conjunction with axial ejection of the ions a t relatively high end-cap rf power as described above for myoglobin. We are currently exploring this possibility. Positive Ion MS/MS and MS" Studies. The behavior of the peptide ions under MS/MS and MS" conditions normally used in the ITMS is obviously of great interest from the standpoint of obtaining structural information from small quantities of material. Indeed, a major motivation behind this work is to evaluate the present ITMS MS/MS operating conditions as they relate to ES-derived ions and to develop new MS/MS methods with the ion trap to maximize structural information obtainable from the ES/ITMS combination. Improvements in mass measurement (see above) must be made before the present MS/MS capabilities of the ITMS for the peptide ions can be thoroughly evaluated. Nevertheless, we have performed some preliminary MS/MS and MS" experiments with some of the peptide cations and present results for melittin to illustrate. By far the most abundant ion derived from melittin consistently observed in this system is the quadruply protonated molecule, (M + 4H)*+,observed at m/z 712.6. Figure 14 shows an MS/MS spectrum of (M + 4H)4+over the mass range in which daughter ions are observed. Roughly 4 pmol of analyte passed out of the capillary needle during the 12-9 period of data acquisition (an average of 10 scans was taken with each scan requiring about 1.2 9). The parent ion mass/charge (712.6) is greater than the nominal mass/charge range of the instrument (650), so that the necessary magnitudes of ring electrode rf amplitude and dc voltage for parent ion isolation over a relatively narrow mass range were not available. A relatively broad mass/charge range could be isolated, however, by using the relevant voltages at or near the maximum values that the electronics could provide. The range of isolated mass/charge values for the data of Figure 14 is about 650-740. Within this range, chemical noise may appear along with
daughter ion signals generated from CID. The daughter ion assignments from m/z 650-740 should therefore be considered to be less reliable than those that fall outside this region. The data were acquired using a low mass/charge cutoff of 250, and the mass/charge range of the instrument was extended by a factor of 6 by using axial modulation. Barinaga et al. (20) have reported triple-quadrupole MS/MS spectra of various charge states of melittin, including that of (M + 4H)4+,produced via electrospray. They list, along with the CID fragments they observed, a variety of possible fragment ions using the terminology of Roepstorff and Fohlman (57). The peaks observed in Figure 14 are labeled by using this terminology (omitting the prime and double prime symbols denoting the transfer of one or two hydrogens, respectively) and are assigned based on the measured mass/charge values. Two labels are associated with a given peak in cases where the mass measurement is insufficient to distinguish between two possible fragments. The qualitative similarity between the spectrum of Figure 14 and the corresponding spectrum acquired by using a triple-quadrupole mass spectrometer (20),over the mass/charge range of 500-1100, is remarkable. Essentially all of the fragment ions observed in this region are common to both spectra, and their relative abundances are similar. This is remarkable in that the conditions under which the MS/MS data are acquired by using the ITMS and the triple-quadrupole instrument are, in many ways, quite different. It is beyond the scope of this paper to detail all of these differences and their possible ramifications. We simply indicate here that there are significant differences in the respective target species (He versus NJ, center-of-mass collision energies, time frames, numbers of collisions possible for the parent ions, numbers (and energies) of collisions possible for daughter ions, and perhaps, instrumental discrimination effects. The major difference between the spectra is that essentially no ions below m / z 500 are observed in the ITMS experiment, whereas a number of singly charged ions were observed in the triplequadrupole study. This is due, in part, to the fact that the ion trap was operated so that no ions below m / z 250 could be observed even if they were formed. Nevertheless, several peaks were clearly observed in the mass/charge range of 250-500 in the triple-quadrupole study and should have been detected if the same ions were formed in the ITMS experiment. This observation may reflect the difference in "multiple-collision conditions" afforded in a beam-type instrument versus those afforded in a trapping instrument (58). More comparisons are necessary, however, to see if this observation proves to be general. As observed in the triple-quadrupole study, the major ions in the mass range of 500-1100 are of the Y type, and most of the Y ions are doubly charged. A signal is attributable to each Y ion from Yloz+ to Ylg2+. Signals can be attributed to singly charged B ions from Bg+to Biz+. Two triply charged Y ions are clearly observed and correspond to Y12+and Ya3+. A discussion of the significance of this spectrum, and those from other charge states, for structural information is given by Barinaga et al. (20) and is not repeated here. However, it is noteworthy that, as with the triple-quadrupole instrument, the MS/MS spectrum obtained with the ITMS does not show conservation of charge. In no case are complementary ions observed in comparable abundance (which stands in contrast to the MS/MS data for the dianions discussed above). Barinaga et al. (20) speculated that charge transfer to the target gas (N,) may be responsible for this observation in the triple quadrupole. Charge transfer to He in the ITMS is highly unlikely. However, air (which includes solvent vapor) is present in the ion trap vacuum system a t a pressure of roughly 1 X Torr, and with the relatively long time frame
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 13, JULY 1, 1990
of the ITMS experiment, many collisions can occur between the ions and background air. However, we typically do not observe significant parent ion loss when time delays of up to several tenths of a second are used just prior to scanning the mass spectrum. This indicates that ion/molecule reactions with background gases do not proceed at significant rates in the absence of kinetic excitation of the parent ion. If charge transfer, or another type of ion/molecule reaction, proceeds when the parent ion is kinetically excited, then the reaction is likely to be endoergic. Unfortunately, it is difficult to excite kinetically the reatively high mass/charge parent ion and trap both high and low mass/charge daughter ions. We, therefore, cannot shed light on this question with the present data. Future MS/MS studies, however, will attempt to address the question of the fate of all of the parent ion charge. The MS/MS spectrum of (M + 4H)4+clearly shows a variety of ions that reflect the structure of the parent ion. Further information may be available from MS" experiments. We have performed such an experiment in which the Y132+ daughter ion formed from CID of (M + 4H)4+is itself subjected to CID (spectrum not shown). Given the poor ion isolation capability for ions at high mass/charge values (Y13'+ = m / z 812), it is difficult to clearly distinguish daughter ions of (M + 4H)4+ from its granddaughters. The only granddaughter ion from (M + 4HI4+clearly apparent from CID of Y12+ falls at m/z 8C3-804 and is formed with a signal intensity of about 40% that of the YI3'+ ion prior to CID. This ion cannot be attributed to any of the possible A, B, C series or X, Y, Z series ions that would be expected from the Y13" ion. Although the mass measurement is too poor to rule out water loss, the most likely interpretation is that the granddaughter ion at m / z 803-804 is doubly charged and is formed by loss of ammonia from Y132+.We have also observed evidence for ammonia loss in several MS3 and MS4 spectra involving ions derived from leucine enkephalin in an extensive MS" study of the protonated molecule (59). Further study is clearly needed, both for singly and multiply charged ions, to evaluate the fragmentation behavior of peptide-derived ions under MS" conditions for structural elucidation purposes. The ES/ITMS combination, with improved ion isolation and mass measurement capabilities, can be expected to be very useful in this regard.
CONCLUSIONS Despite the shortcomings of the present ITMS system for the analysis of ions of mass/charge > 650, it is clear from this work that the combination of ES with a quadrupole ion trap will be extremely useful analytically. The relatively low levels of analyte necessary to obtain good-quality spectra by using the present interface, which may not be optimal, is highly encouraging. Furthermore, the factors that work against the observation of cluster and adduct ions with the present ES/ITMS system may also prove to be advantageous. The ITMS, of course, was not designed to analyze ions of mass/ charge > 650. A few relatively straightforward modifications, such as increasing the number of data points acquired per peak when using axial modulation to extend the mass/charge range, would significantly improve the system. With these improvements and the capability for MS/MS and MS", the ES/ITMS should prove to be extremely attractive due to the information it can provide at a relatively low cost. LITERATURE CITED (1) Blakely, C. R.; McAdams, M. J.; Vestal, M. L. J . Chromatogr. 1978, 158, 264. (2) Blakely, C. R.; Carmody, J. J.; Vestal, M. L. Anal. Chem. 1980. 52,
1636. (3) Blakely, C. R.; Carmody, J. J.; Vestal, M. L. J. Am. Chem. Soc. 1980, 102, 5931. (4) Vestal, M. L. I n Mass Spectromefry in the Health end Life Sciences; Burlingame, A. L., Castagnoli, N., Jr.. Eds.; Elsevier: Amsterdam, 1985; p 99.
(5) Fenn, J. 6.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse. C. M.
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RECEIVED for review January 25,1990. Accepted March 22, 1990. This research was sponsored by the United States Department of Energy Office of Basic Energy Sciences under Contract DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc.
Surface- Induced Dissociation by Fourier Transform Mass Spectrometry Carl F. Ijames and Charles L. Wilkins* Department of Chemistry, University of California, Riverside, California 92521
A detalled procedure for performlng surface-Induced dlssoclatlon ( S I D ) of Ions In a dual-cell Fourier transform mass spectrometer Is described. It Is shown that the technlque Is appllcable to both electron Ionization and laser desorptlon measurements. S I D spectra of perfluorotrl-n-butylamine, anthracene, (5,10,15,20-tetraphenyl-21 H,23H-porphlnato)Iron( I I I ) chloride, and [5,10,15,20-tetrakis( 2,6-dlbromophenyl)-2lH,23HporpMnato]kon( 111) CMOrkle are presented. Converslon efflclencles of molecular Ions between 1 % and 30% are obtained. It Is concluded the method hdds promise for dlssoclatlon of high mass laser-desorbed Ions.
INTRODUCTION A recent and very promising addition to the list of ion fragmentation techniques is surface-induced dissociation (SID),introduced by Cooks and co-workers in 1985 (I). The technique consists of accelerating parent ions into a metal target and then mass analyzing the inelastically scattered daughter ions. In a series of papers, these workers have described implementation of SID using hybrid magnetic sector-quadrupole (I), tandem quadrupole (2), and tandem time-of-flight mass spectrometers (3). These instruments provide collision energies up to 300 eV for electron ionization-generated parent ions and up to unit daughter ion mass resolution. Conversion efficiencies, defined as the sum of daughter ion abundance9 divided by the parent ion abundance in the absence of SID, range from 2% to 15%. It should be noted that this common definition for multiple analyzer instruments exagerates the SID efficiency because it neglects substantial losses of parent ions in the second and subsequent analyzers. The stricter definition, which would use the parent ion abundance arriving at the SID site, gives less than 1% SID efficiency. Here, surface-induced dissociation with comparable efficiency using a Fourier transform mass spectrometer (FTMS), where there is no such distinction, is described. From an analytical standpoint, a major advantage of SID is its ability to convert a relatively high percentage of translational energy into internal energy. For example, 25 eV SID of iron pen0003-2700/90/0362-1295$02.50/0
tacarbonyl ions yields an average internal energy of approximately 4 eV while collisions at the same laboratory energy with argon gas deposit less than 2 eV, on average (4). The average internal energy increases with translational energy, reaching 8 eV at a collision energy of 140 eV. This corresponds approximately to the energy deposited by a 7-keV collision with an argon atom (4). It should also be noted that similar high efficiencies can be obtained under higher pressure, multiple collision conditions (e.g. collision induced dissociation (CID) using a triple quadrupole or ion trap mass spectrometer). A major limitation of conventional collisional activation tandem mass spectrometry (MS/MS) is that the yield of secondary ions decreases as the parent mass increases (5). This is a result of the increasing mismatch in parent and target masses and the greater number of vibrational modes available in the larger molecules, facilitating internal distribution of the deposited energy, rather than dissociation. SID has the potential to increase internal energy deposition and thus to increase the efficiency of MS/MS of high mass ions. Evidence for this is found in Aberth’s recent report of SID of parent ions produced by secondary ion mass spectrometry using a tandem Wien filter instrument (6). A microchannel plate in a grazing incidence configuration was employed as the target and collision energies extended up to lo00 eV. Both positive and negative spectra of leucine-enkephalin (molecular weight 555) were reported, with fragmentation similar to that produced by high-energy CID. Also, (CSI)~&S+was fragmented to yield (CsI),Cs+ ions from n = 0 to 23. These results show that SID is feasible with high mass ions at collision energies as low as 1 keV. Fourier transform mass spectrometry is a technique that offers several advantages over other types of mass analyzers. These advantages, which are well-known, include its extremely high mass resolution, the ability to acquire a complete spectrum following a single pulsed ionization event, and the capability of performing MS/MS analysis without adding additional mass analyzers (7).However, to achieve maximum resolution using FTMS, analyzer cell pressures of less than 1X Torr are required, while pressures of (0.1-1.0) X lo4 Torr are necessary for CID. Also, the maximum cyclotron 0 1990 American Chemical Society
