J. Phys. Chem. 1993,97, 11883-11886
11883
Secondary Electron Analysis of Polymeric Ions Generated by an Electrospray Ion Source Y. Xu, Y. K. Bae, R. J. Beuhler, and L. Friedman' Chemistry Department, Brookhaven National Laboratory, Upton, New York 11 973 Received: June 24, 1993; In Final Form: September 21, 1993'
Electrospray mass spectra of biopolymeric ions can be used to determine ion masses using secondary electron pulse spectra. Secondary electron yields reflect electronic excitation deposited in a solid target, which is related to the electron density and velocity of projectile ions. Secondary electron pulse spectra provide a fingerprint which measures masses of ions of known velocity. Ions with masses from 41 1 (tetraheptylammonium ion) to 480 000 (apoferritin) amu give secondary electron yields that fall on a single curve of secondary electrons/amu plotted as a function of ion velocity. Secondary electron analysis revealed decomposition of larger polyethylene glycol ions ( m > 10 000 amu).
Electrospray ion source techniques which generate multicharged ion spectra1J show great promise as a powerful tool for structural and analytical mass spectrometry of large biomolecules. If electrospraymass spectra show resolved peaks for parent mass ions differing by unit charges, mass determination may be simple and direct. However, in structural studies on purified singlecomponent systems, even qualitative interpretation of unresolved spectra is difficult. Our interest in this problem was stimulated by a search for energetic molecular projectiles that could be used to generate very high energy densities in atomic assemblies in solid surfaces. Multicharged ions could, in principle, be accelerated over relatively short path lengths in small linear accelerators and deliver many megavolts to solid targets. The acceleration of large linear polyethylene glycol (PEG) polymers which could carry hundreds or more charges was a particularly attractive option in our research. But the pioneering work on electrospray of these molecular systems2did not, in our opinion, conclusively establish the charge state of the high molecular weight PEG ions. This is a difficult task with polymer mixtures or spectra of large molecules which show unresolved bands of parent mass multicharged ions. Mass calculation or charge determination of the ions from such spectra requires independent charge determination. We have used secondary electron distributions generated by impact of mass-analyzed ion beams accelerated to 100-200 kV to determine ion masses. Secondary electron yields reflect the extent of electronic excitation deposited in a solid target by ion impact, which in turn is a measure of the mass and velocity of projectile ions.3.4 Thus, the experimental secondary electron distributions combined with calculated ion velocities from mlz values and ion acceleration voltages provide a basis for determination of the charge and mass of the ions. This technique has been developed and established with various small molecular species3 and used for verifying a theoretical model based on stopping power.4 Experimentswerecarried out in theapparatus shown in Figure 1, An Analytica electrosprayion source modified with additional differential pumping was interfacedwith a high molecular weight mass spectrometersystem.' The system consisted of a quadrupole mass analyzer driven by an Extrel 292 kHz power supply with mass analytical capability of 65 000, a 300-kV post-acceleration column, and a secondary electron detection system. Ions generated by electrospraywere mass analyzed by the quadrupole, accelerated to 65-200 kV, and directed to the secondaryelectron detector. The typical pressure of the acceleration column and the chamber containing the detection system was -5 X l e 7 Torr. Abstract published in Advance ACS Absrracrs, November 1, 1993.
The ions were further accelerated by 35 kV in the detection system, after which they struck a copper dynode. Ion currents were limited to ensure low rates on production of secondary electronpulses, i.e. less than ten thousand per second. This served to avoid damage to the solid-state detector and to ensure that the signals observed were produced by isolated ion impact processes. Secondary electrons were accelerated to 35 kV. The magnitude of secondary electron acceleration can be varied from as low as about 20 kV to values limited only by breakdown or by microdischarge phenomena in the detector. Electron acceleration is necessary for producing signals above the noise in the surface barrier detector and for focusing of secondary electrons. Output pulses from the surface barrier detector measure the total energy deposited by the secondary electrons generated during each ion impact. Thus a pulse of 100 electronsaccelerated with an energy of 35 kV will generate a 3.5-MV pulse. The surface barrier detectorsusedin our experimentswere capableof energy resolution of approximately 10-15 kV. With secondaryelectron distributions peaked at between 10 and 20 electrons per pulse, resolution between individual electron peaks in the secondary electron distributions was easily obtainedand the absolutevaluesof energy output from the detector could be calibrated. Alternatively, surface barrier detector outputs can be independently calibrated with a-particle-emitting radionuclides. More details on the use of surface barrier detectors in mass spectrometry can be found in earlier p~blications.3.~ Surface barrier detectors and secondary electron multipliers both have the capability of individual ion detection by measurement of distributions of secondary electrons generated by ion impact. The fact that ion impact generates secondary electron distributions rather than a unique specific number of electrons for an ion of a given mass and energy on a given surface reflects a statistical process that is also operative in the generation of secondary electrons by electron impact. Consequently, the electron multiplication process in electron multpliers, which increases the number of electrons produced by the initial ion impact, also broadens the distribution of electrons. The energy amplification process used in this work preserves the integrity of the initial electron distribution generated by ion impact. Increased sensitivity of detectionof electron pulses is the result of increasing the energy of the electrons rather than their numbers. Both techniques are "surface sensitive" in that secondary electron yields vary with the surfaces from which they are generated. Surface reproducibilitywas maintained in part by limiting integrated ion fluxes. With less than 104 ion/s, dynodes gave reproducible results over many hours of exposure. Clear advantages in calibration and reproducibility result from the use of one dynode surface in the surface barrier detection technique.
0022-365419312097- 1 1883%04.00/0 0 1993 American Chemical Society
11884 The Journal of Physical Chemistry, Vol. 97, No. 46, 1993
Xu et al.
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Figure 2. Secondary electron distributions of various electrospraygenerated ions of biomolecules. The ions were accelerated to 100 kV and struck the copper dynode, which ejected bursts of secondary electrons. The electron yields represent the numbers of electrons simultaneously ejected per ion impact. Part A shows ions of gramicidin S with m = 1 141, m / z 1 141, and z = +l; Part B shows ions of cytochrome c (horse heart) with m = 12 400, m / z 1 550, and z = +8; Part C shows ions of bovine albumin with m = 66 290, m / z 1 381, and z = +48. Note that the peak secondary electron yields are roughly proportional to the masses of the ions.
Figure 3. Secondaryelectron yields per ion impact of various molecular ions as a function of ion velocity. Note that the yield of PEG 100 OOO ions is unusually low.
Secondaryelectron spectra of gramicidin S, cytochromec, and bovine albumin, with molecular weights of 1 141, 12 400, and 66 290, respectively, are presented in Figure 2. The mass spectra of these ions show resolved charge peaks so that the charge state for the ions in each peak can be determined if the adduct ion mass is known. The secondary electron spectra in Figure 2 were obtained by using a multichannel analyzer to plot the number of pulses from the surface barrier detector (the ordinate in Figure 2) as a function of output pulse energy (the abscissa in Figure 2). The pulse energy was translated into the number of electrons per pulse by dividing the energy by the electron accelerating voltage, usually 35 keV. All results wereobtainedwith accelerated ions striking the copper dynode at 100 kV. Because of their similar m / z values, these ions have roughly the same ion velocities. Under this condition, the ions of gramicidin S , cytochrome c, and bovine albumin produced peak yields of 19, 270, and 1 700 secondary electrons, respectively, which are roughly proportional to their molecular weights. Peak secondary electron yields of various ions, some of which gave unresolved spectra,are presented as a function of ion velocity in Figure 3. The molecular weights of tetraheptylammonium, PEG 3 350, cytochromec, bovine albumin, PEG 100 000, gamma
globulin, and apoferritin are 41 1,3 350,12 400,66 290,100 OOO, 160 000, and 480 000, respectively. Both the secondary electron yields at a given velocity and the slopes of plots of yield as a function of velocity systematically increase with the mass of the ions. In Figure 4 the same data are replotted with secondary electrons per dalton as the ordinate. These reduced electron yields for all molecules except PEG 100 000 fall within a narrow band. The open circles represent data for singly charged ions of small water clusters generated by a nozzle ion s o u r ~ e .These ~ data also fall within the band. More precise data can be obtained by calibration of the curve with samples of known molecular weights run under conditions identical to those used for analyzingunknown materials. The curve shown in Figure 4 can be used for calculation of the mass and charge of ions. The following procedure illustrates such a calculation with electrospray-generatedions of apoferritin. A mass spectrum of apoferritin (Figure 5 ) shows two broad bands of mlzvalues peaking at approximately 13 500 and 19 500 without resolved peaks. From these m / z values combined with the acceleration voltage of 185 kV, velocities of these ions corresponding to the two peaks were determined to be 5.2 X 106 and 4.3 X 106 cm/s, respectively. From the curve shown in Figure 4, the reduced secondary electron yields per dalton, 7, for these
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The Journal of Physical Chemistry, Vol. 97, No. 46, 1993 11885 80
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peaks at these velocities were determined to be 4.2 X 10-3 and 2.5 X le3electrons/Da, respectively. The total numbers of electrons per ion impact, I', for these two peaks were estimated from measured secondary electron distributions to be -2 000 and -2 300, respectively. Their respective molecular weights, M,calculated from the relation M = r/y,were -480 000 and -920 000, indicating the peaks are monomers and dimers. The discrepancy between the estimated ratio of the masses of the monomer and dimer masses can be accounted for in part by the uncertainties in the precise measurement of peaks in the respective secondary electron distributions and the peaks of the ion mass to charge ratios. In addition, there is the possibility that solvent removal from the respective species was not as complete in the cases of the "monomer". All things considered,the identification of the bands at m / z 13 500 and 19 500, respectively, as monomer and dimer peaks appears to be reasonable. An independent technique of charge determination is demonstrated in Figure 6. Theopen circles represent the experimental values of peak secondary electron yields of water-methanol cluster ions ( m / z 14 000) generated by the electrospray source. The mass spectra of these clusters did not show any resolved charge peaks; thus it was impossible to determine the mass and charge of the ions directly. Methods described above were used to calculate secondary electron yields for water-methanol cluster ions as a function of ion velocity for three different charge states of 16,21, and 26 (Figure 6). The calculations used secondary electron yields measured with small singly charged water cluster ions of different mass over a range of ion ~elocities.3.~The experimental data fit the curve for 21 charges and establish the cluster molecular weight of -300 000. Note that higher postacceleration voltagesgive the better charge determination, because the curves for ions of different charges diverge rapidly as the ion velocity increases. This clearly demonstratesan advantage of using high-voltagepastacceleration. Secondary electron analysis serves to resolve ions of different mass and charge but with unresolved mass-to-charge ratios. This
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40 MASSKHARGE Figure 5. Electrospray mass spectrum of apoferritin. The signals were measured on the surface barrier detector,and the postaccelerationvoltage which was the sum of the column and detector voltages was 185 kV.The
two peaks do not show any resolved charge peaks; thus the identities of the ions responsible for the peaks are not clear. However, the secondary electron analysis revealed that the two peaks correspond to monomer and dimer ions of apoferritin.
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Figure 6. Secondary electron yields per ion impact of water-methanol cluster ions of m / z 14 OOO generated by the electrospray source as a
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can be done by first observing broadened or resolved secondary electron distributions when ions with a single value of m / z are
Xu et al.
11886 The Journal of Physical Chemistry, Vol. 97, No. 46, 1993
focused on the detector dynode. The secondary electron detection system can then be set up with discriminators which limit the width of thesolid-statedetector energy output so that experimental spectra include only ions capable of generating a narrow band of secondary electrons. In this way ions with different masses and/or different charges can be isolated in different mass spectra. Results obtained with this technique will be presented elsewhere. The difference between the secondary electron yields of PEG 100 000 and other molecules including PEG 3 350 indicates that the PEG 100 000 molecules decomposed into fragments. We have also investigated the mass and charge of electrospraygenerated ions from other PEGs with molecular weights of 20 000, 900 000, and 5 000 000. The mass spectra for these PEGs, except PEG 3 350, showed broad unresolved peaks at m / z 1 200, as observed by Nohmi and Fenn.2 Secondary electron analysis gave masses for these peaks to be 10 000. We conclude that the PEG molecules heavier than 20 000 decomposed into fragments with molecular weights of 10 000. The experiments on PEGs were carried out using 0.001 g/L concentrations of solute similar to thoseused by Nohmi and Fenn2 but with a quadrupole mass analyzer capable of mass analysis of species with m l z up to 65 000. No change in mass spectra was obtainedwith solute concentrations ranging from 0.01-0.001 g/L. The electrospray ion source used was obtained from Analytica, designed by Fenn and co-workers. It was modified to provide for an additional stage of differential pumping. The useof an energy selector between the acceleration column and an off-axis ion detector ruled out PEG decomposition after mass analysis in the acceleration column. We are left with the conclusion that any decomposition that may have taken place must be limited to the relatively short path length between the source of ions and the acceleration column. The mass spectra observed for the PEGs
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were obtained with samples of material kindly provided to us by Professor Fenn. To sum up, the experiments carried out were designed to reproduce the work of Nohmi and Fenn with the addition of a means of identification of the charge on the ions reaching the detector. The question of fragmentation of large molecular species has only surfaced with the long-chain PEG polymers. Electron distributions of the smaller PEG with molecular weight 3 350 appear to be well behaved, as do the biopolymers studied. The possibility that chain structures give rise to secondary electron distributions significantly different from those obtained with molecules that are symmetrical or compact in structure is inconsistent with results obtained with smaller hydrocarbons3 and with the PEG 3 350. We conclude our speculation on the results with PEG polymers by noting that, in addition to the indication of possible limitations in the use of electrospray techniques for structural studies, the goal of generating extremely highly charged polymer ions by use of long-chain polymers which could carry charges in excess of the limits calculated for spherical structures appears to be beyond our present capabilities.
References and Notes (1) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64. 1992, 224, 3241. (2) Nohmi, T.; Fenn, J. B. J. Am. Chem. SOC. (3) Beuhler, R. J.; Friedman, L. Inr. J . MassSpectrom. Ion Phys. 1977, 23, 81. (4) Beuhler, R. J.; Friedman, L. J. Appl. Phys. 1977, 48, 3928. (5) The invaluable technical assistance of S. Howell, V. Chiampou, and G . Borsella is gratefully acknowledged. We also thank Dr. G . Friedlander for stimulating discussions. This research was carried out at Brookhaven National Laboratory under Contract DE-AC02-76CH00016 with the US. Department of Energy and supported by its Division of Chemical Sciences, Office of Basic Energy Sciences and Advanced Energy Projects.
