Anal. Chem. 1994, 66,3964-3969
Direct Charge Number and Molecular Weight Determination of Large Individual Ions by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Ruidan Chen, Qlnyuan Wu, Dale W. Mitchell, Steven A. Hofstadler, Alan L. Rockwood, and Richard D. Smith’ Chemical Sciences Department, Pacific Northwest Laboratory, Richland, Washington 99352
The coupling of electrospray ionization (ESI) techniques with Fourier transform ion cyclotron resonance (FI’ICR) mass spectrometry allows the analysis of individual (Le., single) multiply charged ions. In this paper, we demonstrate that individual large ions can be directly characterized through their excitation and ejection behavior in the FTICR cell. We also report the direct measurement of the charge carried by an individual poly(ethy1ene glycol) ion (5 X lo6nominal molecular weight) and thus obtain the molecular weight of an individual ion (-4.1 X lo6)directly from the mlzmeasurement. These resultsconfirm that the observed ions are indeed large individual molecular ions produced by ESI, as opposed to small fragments, and that an approximate molecular weight can be directly measured on the basis of charge determination and measured mlz. This capability augments the ability for more precise mass determination based upon the observation of a known reaction process (e.g., proton transfer) for individual ions. In less than 20 years since its invention,’ Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has established a unique analytical role because of its ultrahigh resolution and mass measurement accuracy and the ability to interface with various external ion The most significant recent development is likely the combination of electrospray ionization (ESI)7-10 with FTICR,l13l2which has already demonstrated new and unique capabilities for studies of large biopolymer^.'^-'^ Large molecules (Mr > 10 000) are now routinely observable by ESI-MS due to the multiplecharging phenomenon inherent to ESI.l0 The multiple(1) Comisarow, M. B.; Marshall, A. G. Chem. Phys. Lett. 1974, 25, 282-283. (2) Laude, D. A,, Jr.; Hogan, J. D. Tech. Mess. 1990, 57, 155-159. (3) Marshall, A. G.; Schweikhard, L. Int. J. Mass Spectrom. Ion Processes 1992,
11811 19, 37-70. (4) Marshall, A. G.; Grosshans, P. B. Anal. Chem. 1991, 63, 215A-229A. ( 5 ) Fourier Transform Mass Svectrometrv: Evolution. Innovation. and ADplications; Buihanan, M. V.,*Ed.; Amerlcan Chemical Society: Washington, DC, 1987; Vol. 359, p 205. Asamoto, B.;Dunbar, R. C. Analytical Applications of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry; VCH: New York, 1991; p 306. Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 2240-2249. Wong, S.F.; Mann, M.; Fenn, J. B. J . Phys. Chem. 1988, 92, 546-550. Smith, R. D.; LOO,J. A.; Ogorzalek LOO,R. R.; Busman, M.; Udseth, H. R. Mass Spectrom. Rev. 1991, 10, 359-451. Smith, R. D.; LOO,J. A.; Edmonds, C. G.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1990, 62, 882-899. Henry, K. D.; Williams, E. R.; Wang, B.-H.; McLafferty, F. W.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 9075-9078. Henry, K. D.; Quinn, J. P.; McLafferty, F. W. J. Am. Chem. SOC.1991,113, 5447-5449. Senko, M. W.; Beu,S. C.; McLafferty, F. W. Anal. Chem. 1994,66,415417. Beu, S . C.; Senko, M. W.; Quinn, J. P.; McLafferty, F. W. J. Am. Chem. Soc. 1993, 4 , 190-192. Winger, B. E.; Hofstadler, S.A.; Bruce, J. E.; Udseth, H. R.; Smith, R. D. J . Am. Soc. Mass Spectrom. 1993, 4 , 566-577.
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charging phenomenon creates peaks corresponding to Mr/z, as opposed to M,. Interestingly, the “upper mass limit” for the practice of ESI-MS currently results from the failure to resolve individual charge states. This difficulty arises not from limitations to mass spectrometer resolution so much as from the heterogeneity of the material and the resulting overlap of adjacent charge states. Recently, our laboratory reported the trapping and detection of very large molecular ions (poly(ethy1ene glycol), - 5 MDa) by ESI-FTICR mass spectrometry in the m/z = 20003000 range.I6 The observation of individual ions was confirmed by time-resolved ion correlation,I7 in which individual ions were monitored during transient acquisition and observed to undergo stepwise shifts in m/z when reacted with background neutrals. Induced charge state shifts and lower limits of detection for individual ions have also been studied.17 In this paper, we present the results of individual ion studies that used an alternate and complementary approach, in which individual ion analysis methods are performed without the necessity of inducing a change in the mass or charge state of the ion. In a typical ICR experiment, ions are trapped inside an orthorhombic (or cylindrical) cell by applying a dc trapping voltage to each of two opposed trapping electrodes. The trapped ion cloud is then excited to some radius by applying a differential radio frequency voltage on a pair of electrodes which are parallel to the magnetic field but perpendicular to the two trapping electrodes. The image current induced on the two remaining (detection) electrodes by the orbiting ion cloud is converted to a voltage and sampled at equally spaced time increments. The resulting time-domain signal is then Fourier transformed to produce a frequency-domain spectrum, which is in turn converted to an m/z spectrum.18 Theoretically, the peak height is proportional to the number of ions in the cloud, the average cyclotron radius of the ion cloud, the charge number carried by each ion, and the observation interval. For a given ion cloud, the peak height should increase linearly as the cyclotron radius of the cloud increases. When the radius of the given ion cloud is equal to or greater than the cell radius, the ion cloud will be quenched (neutralized) on the electrodes, and thus the corresponding peak height will drop (16) Smith, R. D.; Cheng, X.; Bruce, J . B.; Hofstadler, S. A,; Anderson, G. A. Nature 1994, 369, 137-139. (17) Bruce, J. E.; Cheng, X.; Bakhtiar, R.; Wu, Q; Hofstadler, S.A,; Anderson, G. A.; Smith, R. D. J. Am. Chem. SOC.1994, 116, 7839-7847. (18) Marshall, A. G.; Verdun, F. R. Fourier Transforms in NMR, Optical. and MassSpectrometry: A User’s Handbook; Elsevier: Amsterdam, 1990; p 460. 0003-2700/94/0366-3964$04.50/0
0 1994 American Chemlcal Society
to zero. The corresponding plot of relative peak height vs the ion cyclotron radius (excitation amplitude or excitation time) is called an ion ejection curve. Due to the finite size of the ion cloud, the inhomogeneity of the excitation electric field,19-23 and the ion axial d i ~ t r i b u t i o n , ~there ~ . ~ ~is nearly always a remaining signal (reflecting the low-energy “tail” of the ion cloud) after the expected ejection onset, and the size of such a tail varies with the trapping voltage.26 If there is only one ion responsible for the detected signal, it can be treated as a point chargeand its behavior will bequantized. Theion motion will likely be terminated when it reaches one of the electrodes, and the corresponding peak height will drop to zero. This ejection criterion will be used to distinguish if a peak is due to an individual ion or more ions. A key issue of the individual ion study is whether or not the spectral peaks arise from large (presumably intact) molecular ions. In other words, it must be determined that the ion being monitored is indeed a large molecular ion, as opposed to a number of low molecular weight species or fragments of the molecular ion. Because a mass spectrometer directly measures m/z, molecular weight information cannot be extracted unless the charge state of the ion is also accurately known. The two commonly used methods of charge state determination, which involve analysis of mass spectra resolving a distribution of charge states or resolving the 1 Da isotope spacing of a single charge state, are not applicable to individual ion analysis. Alternative methods for the determination of charge states for individual ions have recently been proposed and demonstrated17 in which a change in the mass and/or charge state of the ion is induced through ion-molecule reactions. Here, we present an alternative approach to charge state determination based on direct measurement. An individual ion produced by ESI can have > 1O3 charges if it is sufficiently large (e.g., >5 X lo6 Da). This number of charges is significantly above the low detection limit of typical ICR instruments. In the detection limit measurements performed by Marshall and c o - w o r k e r ~it, ~was ~ pointed out that the number of charges can be determined with high precision and that “the sensitivity of FTICR-MS for electrosprayed ions approaches single ion detectability”. From the additional ability to directly determine the charge state, the molecular weight can be calculated from a measured m/z. We have found that, under conditions available with our instrumentation, large poly(ethy1ene glycol) (PEG) ions formed by ESI are quite stable, in contrast to the recent report (19) Mordehai, A. V.; Henion, J. D. Rapid Commun. Mass Spectrom. 1992, 6, 345-348. (20) Mitchell, D. W.; Hearn, B. A,; Delong, S . E. Int. J.MassSpectrom. Ion Phys. 1993, 125, 95-126. (21) Hunter, R. L.;Sherman, M.G.; McIver, R. T., Jr. Int. J. MassSpectrom. Ion Phvs. 1983. 50. 259-214. (22) vandeGuchte, W.J.;vanderHart, W. J. Int. J.MassSpectrom. Ionprocesses 1990, 95, 317-326. (23) Huang, S. K.; Rempel, D. L.; Gross,M. L. Int. J.MassSpectrom. Ion Processes 1986. 72. 15-3 1. (24) Chen; R.’ Ph.D. Thesis, The Ohio State University, Columbus, OH, 1993. (25) Rempel, D. L.; Huang, S. K.; Gross, M. L. Int. J. MassSpecfrom.Ion Processes 1986, 70, 163-184. (26) Grosshans, P. B.; Chen, R.; Limbach, P. A.; Marshall, A. G.Int. J. Mass Spectrom. Ion Processes, in press. (27) Limbach, P. A.; Grosshans, P. B.; Marshall, A. G.Anal. Chem. 1993, 65, 135-140.
Quench Pulse Valve
Ion Injection Rear Trapping
-
L
n n
Front Trapplng J
Excitation
L-,
n 1
Detection
-t Figure 1. Experimentalsequence for the generatlon,trapping/coollng, excitation, and detection of ions from a nominal 5 MDa PEG sample producedby the electrosprayprocess.Notethe changes of the trapping voltageduring experiments. Only one quench pulse before ion injectlon is applied to the cell during the entire indlvldual ion experiment.
suggesting that PEGions heavier than -20 000 Da decompose into fragments having molecular weights I 1 0 000.28
EXPERIMENTAL SECTION Experimental Sequences. Due to the size of the molecules studied, the high PEG polydispersity (=l S ) , the large distribution of charge states produced by ESI, and the limited charge capacity of the ICR cell, it is nearly impossible to generate an identical ion population for a series of experiments. The possibility of producing two individual ions of specific mass and charge becomes vanishingly small. Therefore, the observation of the excitation and ejection processes of an individual ion must be encompassed in a single experimental study with the same ion. Thus, in the present studies, an individual ion must be trapped, isolated, and repeatedly remeasured at increasing excitation levels until ejection is accomplished. The optimization of pulse sequences used for ion trapping, cooling, and detection is critical for a successful experiment. Figure 1 shows the experimental sequence for initial ion trapping and detection. The ramping of the trapping voltage on the front trapping plate (the one nearest the ESI source) facilitates the trapping of low kinetic energy ions. Ions are collisionally cooled for 10-20 s at relatively high trapping voltages (5-8 V) before the trapping voltage is dropped to 0.25-0.50 V for excitation and detection. A preliminary excitation and detection step is used to confirm the presence of ions. A relatively low excitation amplitude is typically used for this purpose to allow more rapid ion cooling and remeasurement in the subsequent measurements. There are several distinct differences between the spectra of very highly charged ions ( z >1000) and lower molecular weight multiply charged ions ( z C 100). For instance, it is possible that a specific peak in the mass spectrum of large, highly charged ions arises from two or more ions with very (28) Xu, Y.;Bae, Y.K.; Beuhler, R. J.; Friedman, L. J . Phys. Chem. 1993, 97, 11883-1 1886.
Analytical Chemistry, Vol. 66, No. 22, November 15, 1994
3965
selected m/z. For any orthorhombic cell,
Pulse Valve
3
Rear Traping
Front Trapping
Excitation
I u ' [ u l
h=O
in which f l h is defined depending only on the expansion coefficients for an ion moving in the z = 0 plane. For the elongated cell (aspect ratio a width]:^ [length] = 1:lS) used in our mass spectrometer,
n n
Detection
-t Figure 2. Experimental sequence for the ion cooling, selection (suspended trapping), excitation, and detection. The same sequence is used for ion remeasurements after the removal of the suspended trapping site.
similar m/z values (but with significantly different molecular weights and charge states). Thus, the ions generated by the experimental sequence in Figure 1 are not ideal candidates for individual ion studies. A reduction of the trapped ion population and ion selection is generally needed, which is achieved by suspended t r a p ~ i n g . ' ~Figure ? ~ ~ 2 depicts the experimental sequence used for ion selection. After the initial ion population has been collisionally cooled, the trapping potentials on both trapping plates are grounded for approximately 1 ms. The suspension time can be adjusted to yield an appropriate ion population which, due to the higher velocities of smaller ions, favors retention of high molecular weight ions.16 Following the suspended trapping event and the selection of a smaller and more manageable ion population, multiple remeasurements are performed at increasing excitation levels. The experimental sequence shown in Figure 2 is repeated at increasing excitation levels until the ions are ejected from the trapped ion cell. Charge Number Determination. Marshall and co-worker~2~930 developed the necessary theory to experimentally determine the number of trapped ions, the detection limits, and the dynamic range in FTICR mass spectrometry. The number of ions in the trapped ion cell can be described by27
in which N is the number of singly charged ions in the ICR cell, C is the capacitance of the detection circuit (in farad), Vis the voltage drop in the detection circuit due to the induced image current (in volt), q is the charge carried by one electron (in Coulomb), R is the ion radius normalized to one-half of the cell width (a/2) ,and A ( R ) is defined25.30in the induced differential charge (AQ)equation (neglecting all higher order harmonics) as
A Q = qA(R) cos(w+t) in which o+ is the angular cyclotron frequency for ions at the ~
A(R)=
~~
(29) Laude, D. A., Jr.; Beu, S. C. Anal. Chem. 1989, 61, 2422-2421. (30)Grosshans, P. B.; Shields, P. J.; Marshall, A. 0. J . Chem. Phys. 1991, 94, 5341-5352.
3966 Analytical Chemistty, Vol. 66, No. 22, November 15, 1994
A ( R ) = 0.81402R
+ 0.03051R3 - 0.01369R5 + 0.00200R7
(3) The coefficients of all the expansion terms are numerically evaluated31 and then substituted into eq 2. Since A(R) is used only in the x-y plane, it is somewhat overestimated. Consequently, the resulting N is slightly underestimated. However, the z-dependence of A ( R ) in an elongated cell is smaller than that in a cubic cell since the excitation field is more homogeneous near the center in an elongated cell than in a cubic cell, especially when ion z-motion has been cooled (i.e., reduced) before excitation. Thus, eq 1 can be used to measure the number of charges carried by an individual ion if it is determined that there is only one ion at a given m/z and the charge number is sufficiently large. After measuring the charge state, one obtains the mass of the ion by multiplying the measured charge number by the mass-to-charge ratio of the ion. In order to determine the charge number of an individual ion from the peak height in an acquired spectrum, the capacitance of the detection circuit and the detection circuit response to an induced signal must be well defined. The ion cyclotron radius during detection also needs to be determined for the evaluation of A ( R ) . The details of measurement of detection circuit capacitance, response, and ion radius are discussed in the following section. Instrumentation and Data Processing. The FT-ICR mass spectrometer used in this work was described in detail elsewherei5 and only a brief description is given here. The spectrometer is based on a 7-T superconducting magnet (Oxford) with a 6-in. room temperature bore. The primary ion source is a modified Analytica ESI source (Analytica of Branford, CT). An elongated ICR cell (2 in. X 2 in. X 3 in.) is located in the homogeneous region of the magnet in a vacuum chamber with base pressure 10-lo Torr. Cell control and data manipulation are provided by an IonSpec (Irvine, CA) Omega data station. Ions are transported from the ESI source to the cell by two sets of radio frequency-only quadrupoles. During ion injection, ions are accumulated in the ICR cell by matching the trapping potentials with the kinetic energy (eV/ q ) of the ion beam. Ions are collisionally cooled prior to a swept excitation at 20 Hz/ps (m/z = 1000-5000). The resulting signal is digitized at 500 kHz with 256K data points, resulting in an observation interval of 512 ms. The transient is zero-filled once prior to magnitude-mode Fourier transformation.
-
RESULTS AND DISCUSSIONS Detection Circuit Characterization. The response of the detection circuit was determined by introducing known signals ~~~
~~
(31) Mitchell, D. M.; Rockwood, A. L.; Chen, 1994, in press.
R.;Smith, R. D. J. Chem. Phys.
,
1
0.1 5
-600 1
0.1-
I
0
0 0 0
O
00
200 n
400
0
600
800 L
-200
0
(from 1 pV to 5 mV) from a digital function generator, through a pair of BNC cables, to the detection circuit. Even though the frequency responseof the detection circuit is quite uniform, signals at 55 kHz are used because it is near the frequency of the individual ions observed in this study. The detection circuit is saturated when the input signal is above 800 pV. Below 800 pV, the relationship between the input signal and the FTICR magnitude mode peak height is linear as shown in Figure 3. The calibration equation derived from the data in Figure 3 is relative peak height = 0.0001655 X input signal (pV) (4) Based on this relationship, the voltage drop in the detection circuit induced by an ion (or an ion cloud) can be quickly calculated simply from its corresponding peak height. This voltage drop is then substituted into eq 1 to calculate the charge number, N . The typical voltage drop induced by an individual large PEG ion is approximately 1-2 pV, which is well below the saturation limit of the detection circuit and well above the detection limit. An input voltage of 1 pV yields a signal to noise ratio of -25-30. Calibration of the Detection Circuit Capacitance. In principle, the capacitance of the detection circuit (CO),could be measured directly if the capacitance meter and leads do not introduce additional capacitance. In practice, the calibration is typically performed by adding a known capacitance (C,) in parallel with the detection circuit and monitoring the change of the response of the circuit to a fixed signal input. The general relationship among capacitance (C), charge on a capacitor (Q), and the voltage drop on the capacitor (V) is C=
Q
V
(5)
In this case, C = CO+ C,. Since Vis linearly proportional to the peak height (I)and Q does not change as long as the input signal remains constant, eq 5 may be written as
c, + c, -E -I
1000 2000 3000 4000 5000 6000
or
c,
=
y-co
Therefore, if C, is plotted vs 1/I, the intercept of the plot should yield 40. Figure 4 shows the results of such a measurement. A ubiquitin (Mr = 8565) ion beam from the ESI source is used as the known signal input. The linear correlation coefficient (0.99) of the experimental data demonstrates the satisfactory reproducibility of the source (a more stable signal source might be used to calculate COmore accurately). Themeasured Covalueis 146.1 pF, witha relative error of about 5%, which compares favorably to the estimate of 150 pF obtained using a capacitance meter. The estimate obtained directly from the capacitance meter is subject to additional uncertainty from the internal capacitance of the meter and the test leads. Ejection Curve of an Individual Ion. A unique characteristic of an individual ion, as opposed to an ensemble of ions, is the response to repeated remeasurements at increasing excitation amplitudes. In the ejection curve of an individual ion, the signal intensity increases approximately linearly with increased excitation amplitude ( VPp) and then drops to zero abruptly when the ion impacts on one of the cell surfaces. Typical ejection curves for ensembles of ions demonstrate relatively broad ejection curves owing to such factors as the finite size of the ion cloud, space charge effects?* and z-eje~tion.l~.a"~~J~ In the case of highly charged large molecules, space charge effects will keep ions relatively far away from each other in an ion If a peak is due to two or more ions, its peak height will decrease sharply after the ion ejection onset but will not drop to zero in a single step as it will for the individual ion case. Suspended trapping does not guarantee that each observed peak arises from an individual ion, although the likelihood increases with very small ion populations. Figure 5 demonstrates the difference in ejection curve characteristics between an individual ion and an ion ensemble. An ejection curve such as that shown in Figure 5a supports the contention (32) Jeffries,J.B.;Barlow,S.E.;Dunn,G.H. Int. J. MassSpectrom. IonProcesses 1983, 54, 169-187. (33) vander Hart, W. J.;vandeGuchte, W. J.Int. J.MassSpectrom. IonProcesses 1988,82, 17-31. (34) Beebe-Wang, J.; Elander, N.; Schuch, R. Phys. Scr. 1993, 46, 560-568.
Analytical Chemistry, Vol. 66, No. 22, November 15, 1994
3967
2.0 b)
1.5 -
e e
1.0-
e
e
e
e
0.5 07
15
". v
20
25
30
35
40
Excitation Amplitude (Vp-p) Figure 5. Ejection curves of (a) an individual ion at m/z = 2054 and (b) an ion packet at m/z 3070. The radius of an individual ion is
-
determined by Its ejection curve a. See text for details.
that the measurement of individual ions is feasible using the techniques outlined in the Introduction. In FTICR, for a given ion (or a compact ion cloud), the peak height (signal intensity) is only determined by the number of charges carried by the ion or the total charges in the ion cloud, while the peak position is determined by mass and number of charges, and the mass of the ion is determined by mass-to-charge ratio and charge numbers. Note that the peak height in Figure 5b is smaller than that in Figure 5a, showing that the ions in the ion cloud measured in Figure 5b have total charge significantly lower than that carried by the individual ion measured in Figure 5a. In the case of Figure 5b, it is likely that the ion cloud contains two groups of ions which are separated by some distance and have very close m/z values. At some point during the remeasurement process, the group of lighter ions was ejected, which caused the signal decrease (see the minimum in Figure 5b). After that, the group of heavier ions (then having more charges) was excited to higher radius to give the strong signal (corresponding to the maximum in Figure 5b). Further excitation gradually ejects ions in this group, which is manifested by the tail after the maximum. The ejection experiments were carried out at relatively high excitation amplitudes, as the signal from an individual ion (with 1,500-2000 charges) is usually fairly weak and it is difficult to distinguish signal from noise at small cyclotron radius. Additionally, repeated remeasurements, even at low excitation amplitude, increase the likelihood of ion loss. Additional remeasurements require repeated ion collisional axialization and ion excitation. The former may cause ion loss by magnetron motion expansion; the latter increases the likelihood of axial ion ejection.35 Consequently, a minimal number of remeasurement cycles were used to generate each ejection curve. Determinationof Charge Numbersand Weights of Individual Ions. Before eq 1 is applied to calculate the charge number (35) Chen, R.; Guan, S.;Marshall, A. G. J. Chem. Phys. 1993,100, 2258-2266.
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Analytical Chemistry, Vol. 66, No. 22, November 15, 1994
carried by an individual ion, the ion cyclotron radius must be determined. It is difficult with the present trapped ion cell configuration to measure the ion cyclotron radius satisfactorily as has been done in a linear ICR or by the measurement of the harmonic-to-fundamental ratio.3G39 Fortunately, only the normalized ion cyclotron radius, R, is of importance in this study, not the absolute ion cyclotron radius (see eqs 1 and 2). A very good estimate of R can be derived from the ion ejection onset (see Figure 5a). When V,, = 29.25 V, the observed peak height is at a maximum, and R is assumed to be very close to unity; when V,, = 31 V, the peak height drops to zero, presumably due to radial ion ejection (R = 1). From these data, an approximate R can be estimated as 1 [(31- 29.25)/31] 0.95. Substituting 0.95 into eq 2, A(R) = 0.7903. Substituting this value into eq 1, where C and V have been determined, yields a good estimate of the number of charges responsible for the observed signal, allowing the calculation of the ion's weight. Results from three separate experiments are listed in Table 1. The listed results are for the three largest ions selected from about 15 measurements. In the three different experiments, individual peaks appear at significantly different m/z values. As shown in Table 1, these correspond to different charge states of molecules with similar masses. The relative error of the results is estimated to be about 10%. From eq 1, the relative error of N can be derived as
The first term on the right side of eq 7 can be easily minimized to 51% owing to the inherent precision of the function generator and the detection circuitry utilized. The largest error comes from the determination of C and A(R). C and R carry estimated errors of -5%. Additional error for R may result from the holes in the trapping plates of the cell, perhaps 3%. The total relative error of N can then be calculated from eq 7:
= (ldC/C12
-
+ l(0.81402 + 0.09152R'-
+
0.06845R4 0.0 1400R6) dR/A( R)12)'/'
0.094 in which R = 0.95. The error due to the assumption of z = 0 (the ion has no axial motion) is expected to be negligible due to the long relaxation times (20 s) used prior to excitation and detection. Therefore, the relative error of the final results is about 10%.
-
(36) Limbach, P. A.; Grosshans, P. B.; Marshall, A. G. Int. J . Mass Specfrom.Ion Processes 1993, 123, 41-47. (37) Grosshans, P. B.;Shields, P. J.; Marshall, A. G. J . Am. Chem. SOC.1990,112,
1275-1277.
( 3 8 ) Pan, Y. P.; Ridge, D. P.; Wronka, J.; Rockwood,A. L. Rapid Commun. Mass Spectrom. 1987, I , 121. (39) Nikolaev, E. N.; Gorshkov, M.V.I n t . J . Mass Specfrom.Ion Processes 1985, 64, 115-125.
Table 1. Molecular Welght of Large Indlvldual PEG Ions. m/zb
(peak height)-'
induced signal (FV)
NC
Mr(X106)d
2347 2491 2654
4026 4260 4512
1.501 1.418 1.322
1732 k 173 1637 f 164 1525 f 153
4.07 f 0.41 4.08 f 0.41 4.05 f 0.41
a Relative peak height, 0.000 1655 x input signal (pV). Detection circuit capacitance, 146.1 pF. Mass-to-charge ratio. Charge number carried by the ion. Molecular weight of the molecule.
CONCLUSIONS In this work, the trapping and detection of large individual ions is probed in a novel approach in which the quantized disappearance of a peak is used to determine whether it corresponds to an individual ion. A method for direct charge state determination is presented which is based on the response of a well-characterized detection circuit. This method can be generally used on any ICR instrument and can be a valuable addition to other methods of individual ion a n a l y ~ i s . ~ A 6.~~ distinct advantage of the present approach is that charge and m/z are measured simultaneously. Limited by the cell geometry and other inherent sources of error, the current error of these measurements is estimated to be about 10%. The use of a linear ICR cell can further improve the accuracy of charge determination since the estimate of A ( R ) will be more
-
accurate ( A ( @ is actually equal to R),and has no z-position dependence26 due to the elimination of ion z-ejection. The estimate of ion radius in a linear cell is also more accurate because the excitation and detection processes are both linearized and the relationship between ion radius and excitation power is more ideal. Thus, with the use of a linear ICR cell, a high-sensitivity, low-noise detection circuit and the more accurate measurement of the detection circuit, capacitance, it is conceivable that the error in charge determination, and thus mass, can be reduced to less than 1%. Finally, the present work provides definitive proof that large PEG ions can be produced by ESI, at least under the conditions used in this study, and are not dissociated to form much smaller species, as recently suggested.28
ACKNOWLEDGMENT We thank Drs. Xueheng Chen, Jim Bruce, and Ray Bakhtiar for helpful discussions and the U.S. Department of Energy for support of this research through internal Exploratory Research of Pacific Northwest Laboratory under Contract DE-AC06-76RLO 1830. Pacific Northwest Laboratory is operated by Battelle Memorial Institute. Received for review April 19, 1994. Accepted August 5, 1994." Abstract published in Advance ACS Abmacrs, September 15, 1994.
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