Charge Reduction Electrospray Mass Spectrometry

Anal. Chem. 2000, 72, 52-60

Charge Reduction Electrospray Mass Spectrometry Mark Scalf, Michael S. Westphall, and Lloyd M. Smith*

Department of Chemistry, University of WisconsinsMadison, 1101 University Avenue, Madison, Wisconsin 53706

A new mass spectrometric technique, charge reduction electrospray mass spectrometry (CREMS), allowing the analysis of complex mixtures of biological molecules is described. The charge state of ions produced by electrospray ionization may be reduced in a controlled manner to yield predominantly singly charged ions through reactions with bipolar (i.e., both positively and negatively charged) ions generated using a 210Po r particle source. The electrospray-generated multiply charged ions undergo charge reduction in a “neutralization chamber” positioned before the entrance nozzle to the mass spectrometer. The ions are detected using a commercial orthogonal electrospray time-of-flight mass spectrometer, although the neutralization chamber can be adapted to virtually any mass analyzer. The CREMS results obtained exhibit a signal intensity drop-off with increasing oligonucleotide size similar to that observed with matrix-assisted laser desorption/ionization mass spectrometry. Proton-transfer reactions were found to be responsible for reducing charge on proteins and oligonucleotides in both positive and negative ion mode. In the past decade, the field of mass spectrometry has been revolutionized by two new methods for the analysis of high molecular weight molecules, matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS)1,2 and electrospray ionization mass spectrometry (ESI-MS).3 In MALDI, analyte is combined with a large excess of a simple organic matrix to form a crystalline sample on a probe tip. A brief pulse of laser radiation is absorbed by the matrix, causing it to be vaporized. Analyte molecules are entrained in the resultant gas-phase plume and become ionized in gas-phase proton-transfer reactions. In ESI, buffer containing the analyte is passed through a capillary orifice maintained at a high electric potential. A stream of charged droplets is formed, and subsequent desolvation leads eventually to a stream of charged ions. These sister methods thus both provide relatively gentle means for the production of large ions in the gas phase; once produced, the resultant large ions are amenable to mass spectrometric analysis. The tremendous speed, accuracy, and sensitivity afforded by the new methods have led to their rapid adoption by the biological community and application to a variety of important problems. (1) Karas, M.; Bachman, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1987, 78, 53-68. (2) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (3) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71.

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As powerful as these approaches are, they are not without limitations. In MALDI, the major ion formed is typically the singly charged species. This makes MALDI relatively well-suited to the analysis of mixtures, as a single ionic species is formed from each component in the mixture. However, the ion yield obtained for a given species in MALDI depends on the chemical nature and size of the analyte, as well as varying widely in different regions of the sample.4 In some cases, the conditions of MALDI analysis are also damaging to the analyte.5,6 ESI, on the other hand, samples a flowing stream containing the analyte in a uniform and repeatable fashion and is generally a more gentle ionization method.7 Another advantage of ESI is the ease with which it is combined with on-line liquid-phase separation techniques such as HPLC and capillary electrophoresis7 while MALDI requires offline sample purification. A characteristic of the ESI process is that it produces a variety of charge states for each single analyte species present in the sample.8-11 This characteristic of the method compromises the analysis of complex mixtures, as such mixtures generally yield too many overlapping peaks to permit effective discrimination of the various species present. The ESI analysis of relatively simple mixtures may be accomplished using computer algorithms that transform the multiply charged spectrum to a “zero-charge” spectrum, allowing easier visual interpretation of the spectra.12-17 However, as spectral complexity and chemical noise levels increase, these algorithms are likely to produce artifactual peaks and may miss analyte peaks with low signal intensity.18 An alternative approach to addressing the problem of complex mixture analysis is charge reduction. If (4) Beavis, R. C.; Bridson, J. N. J. Phys. D: Appl. Phys. 1993, 26, 442-447. (5) Zhu, L.; Parr, G. R.; Fitzgerald, M. C.; Nelson, C. M.; Smith, L. M. J. Am. Chem. Soc. 1995, 117, 6048-6056. (6) Nordhoff, E.; et al. J. Mass Spectrom. 1995, 30, 99-112. (7) Smith, R. D.; Loo, J. A.; Ogorzalek Loo, R. R.; Busman, M.; Udseth, H. R. Mass Spectrom. Rev. 1991, 10, 359-451. (8) Loo, J. A.; Udseth, H. R.; Smith, R. D. Rapid Commun. Mass Spectrom. 1988, 2, 207-210. (9) Wong, S. F.; Meng, C. K.; Fenn, J. B. J. Phys. Chem. 1988, 92, 546-550. (10) Covey, T. R.; Bonner, R. F.; Shushan, B. I.; Henion, J. D. Rapid Commun. Mass Spectrom. 1988, 2, 249-256. (11) Meng, C. K.; Mann, M.; Fenn, J. B. Z. Phy. D. 1988, 10, 361-368. (12) Ferrige, A. G.; Seddon, M. J.; Jarvis, S. Rapid Commun. Mass Spectrom. 1991, 5, 374-379. (13) Zhang, Z.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1998, 9, 225-233. (14) Zhou, J. X. G.; Jardine, I. Proceedings of the 38th ASMS Conference on Mass Spectrometry and Allied Topics, June 3-8, 1990; p 134. (15) Reinhold, B. B.; Reinhold, V. N. J. Am. Soc. Mass Spectrom. 1992, 3, 207215. (16) Labowsky, M.; Whitehouse, C.; Fenn, J. B. Rapid Commun. Mass Spectrom. 1993, 7, 71-84. (17) Mann, M.; Meng, C. K.; Fenn, J. B. Anal. Chem. 1989, 61, 1702-1708. (18) Stephenson, J., J. L.; McLuckey, S. A. J. Mass Spectrom. 1998, 33, 664672. 10.1021/ac990878c CCC: $19.00

© 2000 American Chemical Society Published on Web 12/03/1999

it were possible to reduce the charge-state distribution characteristic of ESI, so that predominantly singly charged ions were formed, the result would be a mass spectrometric approach that combined the best points of both MALDI and ESI. It would be gentle, easily coupled with on-line purification, and suitable for mixture analysis. Several approaches have been taken to effect charge reduction in ESI mass spectrometry. Modification of the solution conditions by addition of organic acids or bases to produce ESI ions of mainly a single charge state has been shown.19-21 Another approach merged oppositely charged ions with those produced in ESI at near atmospheric pressure prior to entering the mass spectrometer.22-24 In addition, a series of recently published papers by Stephenson and McLuckey utilized gas-phase reactions between the analyte ions and a singly charged ion of opposite polarity within the vacuum system of an ion trap spectrometer.18,25-33 Altering solution conditions does not allow predictable and controllable manipulation of the charge state for all species present in a given mixture, and although modest reductions in charge were achieved by the second method described above, it was not possible to obtain a population consisting predominantly of singly charged ions. Both the ion trap studies and the approach described here decouple the ionization process from the charge neutralization process. This is beneficial since it allows flexibility in choosing both the sample buffer and the instrumental parameters, conditions that are critical to obtain high-quality electrospray spectra. Separating the ionization and neutralization processes also permits independent control of the degree of charge neutralization. The approach presented here has some further advantages compared to the ion trap studies. Most importantly, the cation or anion used for charge neutralization does not have to be “trapped” with the electrospray ions. This allows charge reduction to be performed over an extremely wide range of m/z ions, independent of the neutralizing cation or anion’s m/z value. Conversely, the restricted m/z window characteristic of ion traps limits the analyte m/z range accessible, based on the size of a given neutralizing ion.29,30,32 In addition, since the technique described here does not require a specific anionic or cationic species, switching (19) Mirza, U. A.; Chait, B. T. Anal. Chem. 1994, 66, 2898-2904. (20) Muddiman, D. C.; Cheng, X.; Udseth, H. R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1996, 7, 697-706. (21) Cheng, X.; Gale, D. C.; Udseth, H. R.; Smith, R. D. Anal. Chem. 1995, 67, 586-593. (22) Ogorzalek Loo, R. R.; Udseth, H. R.; Smith, R. D. J. Phy. Chem. 1991, 95, 6412-6415. (23) Ogorzalek Loo, R. R.; Udseth, H. R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1992, 3, 695-705. (24) Griffey, R. H.; Sasmor, H.; Greig, M. J. J. Am. Soc. Mass Spectrom. 1997, 8, 155-160. (25) McLuckey, S. A.; Stephenson, J., J. L.; Asano, K. G. Anal. Chem. 1998, 70, 1198-1202. (26) Stephenson, J., J. L.; McLuckey, S. A. J. Am. Chem. Soc. 1996, 118, 73907397. (27) Stephenson, J., J. L.; McLuckey, S. A. Anal. Chem. 1998, 70, 3533-3544. (28) Stephenson, J., J. L.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 1998, 9, 957-965. (29) Stephenson, J., J. L.; McLuckey, S. A. Int. J. Mass Spectrom. Ion Processes 1997, 165/166, 419-431. (30) Stephenson, J., J. L.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 1998, 9, 585-596. (31) Stephenson, J., J. L.; Van Berkel, G. J.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 1997, 8, 637-644. (32) Stephenson, J., J. L.; McLuckey, S. A. Rapid Commun. Mass Spectrom. 1997, 11, 875-880. (33) Stephenson, J., J. L.; McLuckey, S. A. Anal. Chem. 1996, 68, 4026-4032.

between positive and negative modes of electrospray is straightforward. This allows, for example, protein cations to be neutralized in positive ion mode or DNA anions to be neutralized in negative ion mode without having to change any instrumental conditions other than the operating polarity. Finally, the approach described here is readily implemented by a simple modification to an existing ESI source and is thus adaptable to virtually any ESI instrument. However, the m/z of typical charge-reduced biopolymers will quickly exceed the present upper limit for conventional mass analyzers other than time-of-flight (TOF). This makes charge reduction electrospray mass spectrometry (CREMS) most suitable for use with ES-TOF systems. The approach presented here for charge reduction is fairly wellestablished in the field of aerosol analysis.34-46 R Particles produced by a radioisotopic source such as 241Am or 210Po produce a variety of both positively and negatively charged ions (bipolar ions) from a bath gas containing the elements carbon, nitrogen, and oxygen. The resultant ions can react with and neutralize other ionic species (i.e., multiply charged analyte molecules from ES ionization). We explored this effect in a recent study,47 in which oligodeoxynucleotide ions were produced by ESI, reduced in charge using a 210Po source, and analyzed by differential mobility analysis (DMA). This study demonstrated the efficacy of the charge neutralization, although the resolutions of the DMA spectra obtained were too low to be generally useful for biomolecular analysis. Subsequently, we published a brief report showing the use of charge neutralization in conjunction with ESI-MS.48 In the present work, these results are extended to the analysis of more complex mixtures and the neutralization mechanism in CREMS is studied in some detail. It is shown that proton-transfer reactions mediate the charge reduction process, and the ability of CREMS to dramatically reduce charge-state distributions obtained from DNA and protein samples is demonstrated. EXPERIMENTAL SECTION All protein samples were obtained from Sigma Chemical Co. (St. Louis, MO) and used without further purification. For positive mode analysis, protein samples were diluted in a buffer of 1:1 (v/ v) H2O/CH3CN, 1% (v/v) acetic acid to a concentration of 5 µM. For negative mode analysis, protein samples were diluted in a (34) Chen, D. R.; Pui, D. Y. H.; Kaufman, S. L. J. Aerosol Sci. 1995, 26, 963977. (35) Kaufman, S. L.; Zarrin, F.; Dorman, F. D. United States Patent 5,247,842, 1993. (36) Kaufman, S. L.; Skogen, J. W.; Dorman, F. D.; Zarrin, F.; Lewis, K. C. Anal. Chem. 1996, 68, 1895-1904. (37) Kaufman, S. L. J. Aerosol Sci. 1998, 29, 537-552. (38) Zarrin, F.; Kaufman, S. L.; Dorman, F. D. United States Patent 5,076,097, 1991. (39) Lewis, K. C.; Dohmeier, D. M.; Jorgenson, J. W.; Kaufman, S. L.; Zarrin, F.; Dorman, F. D. Anal. Chem. 1994, 66, 2285-2292. (40) Lewis, K. C.; Jorgenson, J. W.; Kaufman, S. L. J. Capillary Electrophor. 1996, 3, 229-234. (41) Fuchs, N. A. Geofis. Pura Appl. 1963, 56, 185-193. (42) Liu, B. Y. H.; Pui, D. Y. H. J. Colloid Interface Sci. 1974, 49, 305-312. (43) Liu, B. Y. H.; Pui, D. Y. H. J. Colloid Interface Sci. 1974, 47, 155-171. (44) Liu, B. Y. H.; Pui, D. Y. H. Aerosol Sci. 1974, 5, 465-472. (45) Wiedensohler, A. J. Aerosol Sci. 1988, 19, 387-389. (46) Whitby, K. T.; Liu, B. Y. H. Atmos. Environ. 1968, 2, 103-116. (47) Mouradian, S.; Skogen, J. W.; Dorman, F. D.; Zarrin, F.; Kaufman, S. L.; Smith, L. M. Anal. Chem. 1997, 69, 919-925. (48) Scalf, M.; Westphall, M. S.; Krause, J.; Kaufman, S. L.; Smith, L. M. Science 1999, 283, 194-197.

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Figure 1. Schematic diagram of the ESI source and charge neutralization chamber.

buffer of 1:1 (v/v) H2O/CH3 OH, 1% (v/v) ammonium hydroxide to a concentration of 5 µM. All oligodeoxynucleotides (seven oligomers ranging from 15 to 51nt in length) were obtained reversed-phase HPLC purified from Integrated DNA Technologies, Inc. (Coralville, IA) and diluted in a buffer of 1:1 H2O/MeOH, 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (adjusted to pH 7 with triethylamine)49 to a concentration of 5 µM. This HFIP buffer was the best of those evaluated for suppressing Na+ and K+ adduct formation in CREMS analysis of DNA. The 51 mer mixed-base oligonucleotide sequence is as follows: 5′ (TGT AAA ACG ACG GCC AAG CTT GCA TGC CTG CAG GTC GAC TCT AGA) 3′. All other oligonucleotide sequences (15, 21, 27, 33, 39, and 45 mer) are identical to the 51 mer sequence, beginning at the 5′ end and ending at the indicated length. The instrument (Figure 1) is composed of three basic components: (i) a positive pressure ESI source; (ii) a charge neutralization chamber; and (iii) an orthogonal TOF mass spectrometer. The ESI source consists of a 24 cm long fused-silica polyimide-coated capillary (150 µm o.d., 25 µm i.d.) with the spray end conically ground to a cone angle (angle between capillary axis and cone surface) of 35° (No. 2001145, Polymicro Technologies Inc., Phoenix, AZ). The inlet of the capillary is immersed in analyte solution contained in a 0.5 mL polypropylene PCR tube. A positive pressure of 7 psi (49 kPa) is applied to the sample container to produce a typical flow rate of 0.13 µL/min. The solution is maintained at a potential of 4500 V (positive, for positive ion mode or negative, for negative ion mode) by means of a platinum electrode immersed in the sample. The spray is stabilized (49) Apffel, A.; Chakel, J. A.; Fischer, S.; Lichtenwalter, K.; Hancock, W. S. Anal. Chem. 1997, 69, 1320-1325.

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against corona discharge50,51 with a sheath gas of CO2 (1 L/min) fed through a stainless steel tube (1.5 mm i.d.) concentric with the silica capillary. The ESI-generated aerosol containing analyte is carried into the neutralization chamber by a bath gas of medical air (4 L/min) flowing through the spray manifold (Figure 1). The neutralization chamber consists of an aluminum cylinder with a diameter of 1.9 cm and a length of 4.3 cm that is insulated from the spray tip with a Teflon coating. There is a 3.1 cm diameter hole cut into the cylinder and casing in which a 5 mCi 210Po R particle source (No. P-2042, NRD Inc., Grand Island, NY) sits. The R particles react with components of the bath and sheath gases to form a bipolar neutralizing gas, and subsequent reactions between the bipolar ions and aerosol droplets and/or analyte ions lead to neutralization.34-46 The charge-reduced aerosol exits the neutralization chamber through a 3 mm diameter outlet held equipotential with the entrance nozzle of the mass spectrometer. A portion of the aerosol is swept into the entrance nozzle, and the ions pass first through a skimmer and next an rf-only quadrupole, and are finally pulsed down the flight tube, and detected with a microchannel plate detector. The orthogonal TOF mass spectrometer (PerSeptive Biosystems Mariner Biospectrometry workstation) employed for these studies was chosen on the basis of the greater m/z range accessible with this instrument configuration than with other electrospray mass analyzer designs. The Mariner has a preset m/z range of 25 000 amu (based on timing considerations) with (50) Ikonomou, M. G.; Blades, A. T.; Kebarle, P. J. Am. Soc. Mass Spectrom. 1991, 2, 497-505. (51) Zeleny, J. Proc. Cambridge Philos. Soc. 1915, 18, 71-79.

Figure 2. Charge-state reduction of ubiquitin (5 µM in 1:1 H2O/CH3CN, 1% acetic acid) as a function of exposed area of the source: (a) 0 (control), (b) 7.5, (c) 17.5, (d) 27.5, (e) 47.5, and (f) 100% exposed.

a measured external mass accuracy of better than 100 ppm (using angiotensin, 432.9 amu, as a standard). The original electrospray chamber of the instrument was replaced with the ESI source and neutralization chamber described above. RESULTS AND DISCUSSION Two factors are important in determining the degree of charge reduction occurring in the neutralization chamber: (i) the R particle flux from the radioactive source and (ii) the residence time of the aerosol particles in the neutralization chamber. The R particle concentration in the chamber is modulated in these studies by placing a thin brass disk (0.005 in. thick), with a variable number of 2 mm diameter holes drilled in it, between the 210Po source and the neutralization chamber. The source is completely shielded by a brass disk with no holes and shielded proportionally to the exposed surface area when holes are present in the disk. The residence time of the aerosol particles is controlled by varying the flow rate of the bath gas which sweeps the particles through the neutralization chamber. A lower flow rate of bath gas allows longer residence time and more extensive neutralization; a higher flow rate leads to shorter residence time and produces less charge reduction. By balancing the residence time with the R particle source exposure, a charge distribution characteristic of a “neutral” aerosol34-46 may be obtained. It was determined experimentally that with the instrument configuration described, varying exposure to the R particle source provides better control of the chargestate reduction than does changing the residence time in the chamber. Figure 2 shows a series of positive ion mass spectra for a single protein (ubiquitin, 8564.8 amu) with increasing R particle flux.

210Po

R particle

The residence time of the particles in the neutralization chamber was held constant with a constant flow rate of bath gas (4 L/min). The mass spectra shown were obtained from a 5 µM sample over a period of 250 s at a flow rate of 0.13 µL/min. A total of 25 spectra were summed, consuming 0.54 µL (2.7 pmol) of sample. With the 210 Po R particle source completely occluded (Figure 2a), a typical ESI charge distribution was observed, exhibiting six major charge states (+7 to +2) with the +5 charge state as the peak of the distribution. Also evident are many peaks in the low-m/z region (see below). As the R particle flux was increased (exposure of 210Po increased, Figure 2b-e), the charge-state distribution shifted toward lower charge (higher m/z) states. Finally, with the 210Po source completely unshielded (Figure 2f), only two charge states were evident, with the major peak corresponding to the +1 charge state. The unlabeled, low-m/z peaks evident in Figure 2a and b have isotopic distributions and m/z values corresponding to predominantly singly charged and some multiply charged ions that match those expected for ubiquitin fragments. These fragment peaks can be attributed to collision-activated dissociation (CAD) in the nozzle-skimmer (N-S) region of the mass spectrometer.52 The fragment peaks disappear in Figure 2c-f because, at a given N-S potential, multiply charged ions are more prone to dissociation in the N-S region than are ions of lower charge states.53 Therefore, as charge reduction lowers the charge state, the propensity for fragmention also decreases. For this set of experi(52) Smith, R. D.; Loo, J. A.; Baringa, C. J.; Edmonds, C. G.; Udseth, H. R. J. Am. Soc. Mass Spectrom. 1990, 1, 53-65. (53) Loo, J. A.; Udseth, H. R.; Smith, R. D. Rapid Commun. Mass Spectrom. 1988, 2, 207-210.


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Figure 3. Conventional ESI spectrum of an equimolar mixture of seven proteins (5 µM each in 1:1 H2O/CH3CN, 1% acetic acid): neurotensin, melittin, glucagon, insulin, ubiquitin, cytochrome c, and myoglobin.

Figure 4. The same equimolar seven-component protein mixture from Figure 3 (a) without charge reduction and (b) with charge reduction. Both spectra are plotted on the same m/z scale.

ments, the nozzle potential was held at 450 V (the skimmer is held at a constant 16 V for all experiments). In subsequent experiments, the nozzle potential was dropped to 250 V to alleviate CAD of multiply charged ions when charge reduction was not 56 Analytical Chemistry, Vol. 72, No. 1, January 1, 2000

employed. When charge reduction was employed, the nozzle voltage was held at 450 V because the higher nozzle potential was observed to both increase signal intensity and decrease the amount of adduction observed for the lower charge-state ions.

Figure 5. Positive mode analysis of an equimolar mixture of seven oligonucleotides (5 µM in 1:1 H2O/CH3OH, 400 mM HFIP (adjusted to pH 7 with triethylamine)49 (a) without charge reduction and (b) with charge reduction. Both spectra are plotted on the same m/z scale. The inset shows an expanded view of (a) without charge reduction. The spectra were smoothed by convolution with a Gaussian function using software supplied with the spectrometer.

The conventional ESI spectrum of an equimolar, sevencomponent protein mixture (neurotensin, 1672.9 amu; melittin, 2847.5 amu; glucagon, 3482.8 amu; bovine insulin, 5736.6 amu; bovine ubiquitin, 8564.8 amu; equine cytochrome c, 12 360 amu; apomyoglobin, 16 951 amu) is shown in Figure 3. Because the identity and mass of each protein in the mixture is known, it is possible to label each of the 34 peaks corresponding to the various charge states of the analytes in this complicated spectrum. Figure 4a shows this same ESI spectrum compared to the CREMS analysis (Figure 4b) of the protein mixture, both on the same expanded m/z scale. The greatly simplified CREMS spectrum consists of only the singly and some doubly charged peaks for each of the seven analytes. A characteristic of charge reduction evident in Figures 2 and 4 is the decrease in signal intensity with increased charge reduction (compare scales of vertical axes). This loss of signal is expected since the charge reduction process necessarily converts many of the ions to neutrals, which are not detected. The mass resolutions of the spectra are unaffected by the charge reduction, with mass resolutions of about 1000 with or without charge reduction. Further study will be required to determine the change in detectability brought about by charge reduction. Future improvements in the source and spectrometer design, including improved ion optics, may help to mitigate the signal loss brought about by charge reduction. The effect of charge reduction on a mixture of oligonucleotides is illustrated in Figures 5 (positive ion mode) and 6 (negative ion mode). An equimolar mixture of seven oligonucleotides (15 mer,

4586.1 amu; 21 mer, 6440.3 amu; 27 mer, 8293.5 amu; 33 mer, 10 147 amu; 39 mer, 12 002 amu; 45 mer, 13 856 amu; 51 mer, 15 709 amu) was prepared and analyzed with and without charge reduction in both positive and negative ion modes. The mass spectra shown were obtained from a 5 µM sample over a period of 250 s at a flow rate of 0.13 µL/min. A total of 25 spectra were summed, consuming 0.54 µL (2.7 pmol) of sample. In positive ion mode without charge reduction (Figure 5a), a complex spectrum was obtained corresponding to several different charge states for each oligonucleotide as well as other peaks due to base loss (see below). In positive ion mode with charge reduction (Figure 5b), a simple spectrum corresponding to the singly and some doubly charged (unlabeled) peaks for each oligonucleotide was produced. In negative ion mode without charge reduction (Figure 6a), the spectrum resembles that of Figure 5a in complexity, although a greater number of charge states at lower m/z predominate for each analyte and there is a decrease in both base loss and adduction and an increase in signal intensity. In negative ion mode with charge reduction (Figure 6b), the spectrum closely resembles that of Figure 5b, with roughly equivalent intensity, although again with a lesser degree of adduction for the negative mode spectrum. Several aspects of Figures 5 and 6 are worthy of comment. Although the mixtures consist of equimolar amounts of each of the seven oligonucleotides, a precipitous drop in signal intensity for the larger oligonucleotides is evident in both the positive and negative ion mode charge reduction spectra. This general loss of signal with increasing size is also seen in the MALDI-TOF analysis Analytical Chemistry, Vol. 72, No. 1, January 1, 2000

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Figure 6. Negative mode analysis of the same seven-component equimolar mixture oligonucleotides from Figure 5 (a) without charge reduction and (b) with charge reduction. Both spectra are plotted on the same m/z scale. The inset shows an expanded view of (a) without charge reduction. The spectra were smoothed by convolution with a Gaussian function using software supplied with the spectrometer.

of oligonucleotides.54-58 A number of possible explanations exist for this behavior shared by these two very different modes of ionization, including the following: a similar instrumental and/ or detector bias toward smaller oligonucleotides (note that in the present work both methods employ time-of-flight detection systems); increased Na+ and K+ adduction of larger oligonucleotides, resulting in a dividing of their signal over many peaks; a similarity between the two ionization mechanisms favoring the ionization of smaller oligonucleotides;59,60 and/or a difference in the collisional cross sections61-64 between larger and smaller oligonucleotides allowing a greater percentage of the smaller oligonucleotides to reach the detector. Integration of the visible adduct peaks does not completely account for the signal intensity loss for larger oligonucleotides. It is interesting that a similar falloff (54) Mouradian, S.; Rank, D. R.; Smith, L. M. Rapid Commun. Mass Spectrom. 1996, 10, 1475-1478. (55) Nordhoff, E. Trends Anal. Chem. 1996, 15, 240-250. (56) Wu, K. J.; Shaler, T. A.; Becker, C. H. Anal. Chem. 1994, 66, 1637-1645. (57) Fitzgerald, M. C.; Zhu, L.; Smith, L. M. Rapid Commun. Mass Spectrom. 1993, 7, 895-897. (58) Murray, K. K. J. Mass Spectrom. 1996, 31, 1203-1215. (59) Schlag, E. W.; Levine, R. D. J. Phys. Chem. 1992, 96, 10608-10616. (60) Schlag, E. W.; Grotenmeyer, J.; Levine, R. D. Chem. Phys. Lett. 1992, 6, 521-527. (61) Hudgins, R. R.; Mao, Y.; Ratner, M. A.; Jarrold, M. F. Biophys. J. 1999, 76, 1591-1597. (62) Clemmer, D. E.; Jarrold, M. F. J. Mass Spectrom. 1997, 32, 577-592. (63) Shelimov, K. B.; Clemmer, D. E.; Hudgins, R. R.; Jarrold, M. F. J. Am. Chem. Soc. 1997, 119, 2240-2248. (64) von Helden, G.; Wyttenbach, T.; Bowers, M. T. Science 1995, 267, 1483.


in signal is not evident in the protein mixtures analyzed; this suggests that the chemistry of the analytes may play a role in the falloff, possibly with regard to ionization efficiency and/or ion stability. Further work will be required to elucidate the cause(s) for the signal drop-off seen with the MALDI and CREMS analysis of DNA. As mentioned above, there is a greater degree of base loss observed in the normal ESI positive mode analysis of DNA than is observed in the negative mode (see insets of Figures 5a and 6a). A similar effect is observed in the MALDI analyis of DNA and has been attributed in that case to a lower intrinsic stability of the positively charged DNA ions.65 The absence of base loss in either the positive or negative ion mode CREMS spectra indicates that both positive and negative singly charged DNA ions are stable under the conditions employed. It should be noted that the low-mass peaks present in the negative mode, non-chargereduced spectra (Figure 6a and inset) arise from the HFIP buffer and are not observed with charge reduction either in the negative ion mode or in the positive ion mode. The charge-reduced spectra for both the positive and negative mode oligonucleotide mixtures (Figures 5b and 6b) show roughly the same signal intensity, although the positive mode spectrum displays a greater degree of adduction. These results demonstrate that CREMS is an equally useful charge reduction technique in both positive and negative ion modes. (65) Tang, W.; Nelson, C. M.; Zhu, L.; Smith, L. M. J. Am. Soc. Mass Spectrom. 1997, 8, 218-224.

Figure 7. Plot of mass versus charge state showing proton transfer as the mode of charge neutralization for (a) positive mode and (b) negative mode analysis of ubiquitin with variable exposure of the 210Po R particle source. Panel a uses the data from Figure 2.

To characterize the reactions occurring between the multiply charged analyte ions and the bipolar ions produced by the 210Po R particle source, Figures 7 and 8 show plots of the mass versus charge state for positive and negative mode analysis of the protein ubiquitin (Figure 7) and a 33 mer oligonucleotide (Figure 8). To calculate the mass of a multiply charged ion, m/z is multiplied by the charge state, but no correction is made for possible protonation/deprotonation of the ion. The e- theoretical line is calculated assuming only electron-transfer reactions, and the H+ theoretical curve is calculated by assuming only protonation (positive mode) or deprotonation (negative mode) reactions. The values of m/z for both ubiquitin and the 33 mer were calculated for all positive ion spectra by calibration against a multicomponent, positive ion external mass calibration mixture and for all negative ion spectra by calibration against a multicomponent, negative ion external mass calibration mixture. Figure 7a shows that protonation is the sole ionizing reaction taking place for the positive mode analysis of ubiquitin regardless of the amount of R particle source exposure. Figure 7b likewise shows no difference due to degree of R particle exposure and deprotonation as the only ionization mechanism for analysis of the protein in the negative mode. Figure 8a shows only protonation in the positive mode analysis of a 33 mer oligonucleotide regardless of the amount of R particle source exposure. Figure 8b shows deprotonation for any amount of exposure of the R particle source in negative ion mode analyis of the 33 mer oligonucleotide.

Figures 7 and 8 demonstrate that neutralization reactions occurring between the bipolar ions and the multiply charged analytes are exclusively proton-transfer reactions. This aspect of CREMS is beneficial since only a single reaction mediates the charge state of analyte ions. Multiple distinct ionization processes could lead to multiple peaks for each analyte, resulting in undesirably complicated spectra. A discussion of possible ionization processes and reagent ions in atmospheric pressure ionization mass spectrometry, which occurs under conditions similar to those utilized in the CREMS process, may be found in Carroll et al.66 Further study will be needed to determine the specific reagent ions and reaction pathways involved in the CREMS process. CONCLUSIONS Charge reduction electrospray mass spectrometry combines some of the strengths of both MALDI and ESI for the analysis of complex mixtures of both proteins and DNA. Through protontransfer reactions with a bipolar ionizing gas, highly charged ions produced by electrospray ionization (either positive or negative) can be reduced to predominantly the singly charged species. The charge neutralization chamber, used here in conjunction with a commercial ES-TOF instrument, can be adapted to virtually any mass analyzer. However, with charge reduction, the relatively high mass of common proteins and nucleic acids can quickly exceed (66) Carroll, D. I.; Dzidic, I.; Horning, E. C.; Stillwell, R. N. Appl. Spectrosc. Rev. 1981, 17 (3), 337-406.


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Figure 8. Plot of mass versus charge state showing proton transfer as the mode of charge neutralization for (a) positive mode and (b) negative mode analysis of a 33 mer oligonucleotide with variable exposure of the 210Po R particle source.

the m/z range accessible with most instruments. An orthogonalTOF system such as that employed here is clearly the best design presently available for such applications, due to the very high intrinsic m/z range of TOF analyzers. The same drop-off in signal intensity with increased size of oligonucleotides that is seen with MALDI analysis is also observed with CREMS. Discovering the cause of this signal drop-off in the mass spectral analysis of oligonucleotides is a major focus of current research in our laboratory; remedying it has potential for substantially extending accessible mass range in the CREMS and possibly MALDI analysis of nucleic acid mixtures.


ACKNOWLEDGMENT Supported by Department of Energy Grant DE-FG02-91ER61130, NIH Grants R01HG00321 and R01HG001808, and NIH-GMS 5T32GMO8349 training grant (M.S.). We thank Drs. Scott McLuckey and Henry Benner for helpful comments and suggestions.

Received for review August 4, 1999. Accepted October 27, 1999. AC990878C

Charge Reduction Electrospray Mass Spectrometry

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