Anal. Chem. 1995,67, 586-593
Charge State Reduction of Oligonucleotide Negative Ions from Electrospray ionization Xueheng Cheng, David C. Gale, Harold R. Udseth, and Richard D. W i t h * Chemical Sciences Department, Pacific Northwest Laboratory, Richland, Washington 99352
We have investigated the feasibility of simplifying the electrospray ionization ( S I ) mass spectra for mixture analyses through charge state reduction. Two methods for charge state reduction of gas phase oligonucleotide negative ions were evaluated (1) the addition of acids to the oligonucleotide solution and (2) the formation of diamine adducts followed by dissociation in the interface region. In the first method, the efticiency of charge state reduction depends on the pKa, the concentration, and the nature of the acids. Acetic and formic acids were found to be better reagents than HCl, CF3CO& and bPO4. The second method has the advantage that the stability of oligonucleotides is not affected but q u i r e s the optimization of the interface dissociation conditions and the amounts of diamine added to the oligonucleotidesolution. Both methods show promise for charge state reduction, and results are presented for two oligonucleotides: d(p’l312 and d(AGCT). Substantial reduction in spectral complexity upon charge state reduction was also observed for a four-componentmixture of oligonucleotides. Electrospray ionization (ESI) is one of the most important ionization techniques for biopolymers due to its compatibility with solutions of near physiological composition and its high efficiency for production of intact (pseudo)molecular For large molecules, ESI generally produces ions with a broad distribution of charge states having relatively low mass-tocharge ratios (m/z < 2500). This allows the use of conventional, limited m/z range mass spectrometersto analyze samples of high molecular weights. The multiple charging, however, also increases the complexity of the spectra and may concomitantly decrease dynamic range, reduce sensitivity, and compromise mixture component analysis. Mass spectrometers based on ion trapping, such as the Fourier transform ion cyclotron resonance (FTICR) or electrodynamic (e.g., quadrupole) ion traps, are particularly affected because of their limited storage capacity for ions due to space charge effects. Other types of mass spectrometers are also affected due to the fact that the ESI process produces an ion current that is relatively constant (1 x 10-7-5 x A at atmospheric pressure) under typical condition^.^.^,^-* The extensive multiple charging decreases (1) Fenn, J. B.; Mann, M.; Meng, C. IC;W o w , S. F.; Whitehouse, C. M. Science 1989,246, 64-71. (2) Fenn, J. B.; Mann, M.; Meng, C. K; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990,9, 37-70. (3) Smith, R D.; Loo, J. A; Edmonds, C. G.; Barinaga, C. J.; Udseth, H. R Anal. Chem. 1990,62,882-899. (4) Smith, R D.; Loo, J. A; Loo, R R 0.; Busman, M.; Udseth, H. R Mass Spectrom. Rev. 1991,IO, 359-451. (5) Covey, T. R; Bonner, R F.; Shushan, B. I.; Henion, J. Rapid Commun. Mass Spectrom. 1988,2, 249-256.
586 Analytical Chemistry, Vol. 67, No. 3, February 1, 1995
the ion number obtainable for any particular isotope peak of a particular charge state. For a mixture, the convolution of these charge state distributions over the distribution of many components generally yields an uninterpretable mass spectrum due to a lack of sufficient resolution and sensitivity (i.e., too few molecular ions distributed over too many possible charge states/isotope peaks). Thus there have been few studies on the use of ESI for the analysis of more complex mixtures?JO If the charge states of ions from ESI can be compressed into a more limited number, however, the dynamic range and sensitivity will be enhanced, the spectra will be simplXed, and the analysis of more complex mixtures will become possible. We have investigated the feasibility of compressing the distribution of charge states by shifting them to lower charge values (to higher m/z, Le., charge state reduction). In addition to the nature and number of charge-bearing groups in the molecules of there are a variety of other factors that affect the charge state distribution due to the complexity of the ESI process and instrumentation (including solution pH,11-13the nature of cosolutes and solvents,14interface voltage gradient,15J6 interface capillary temperature and desolvation gases,17J8among others). Many of these factors are interrelated. For example, changes in the solution pH, solvents, and heating conditions can all change the conformation of proteins and affect the charge state distribution. However, these complex factors also present opportunities for charge state reduction by a judicious choice of the various ESI conditions. We identify three different stages of the ESI process, each of which can be manipulated independently or together for charge state reduction: solution composition, interface conditions, and gas phase processes. In this article, we report the results of our investigation of two methods for charge state reduction of oligonucleotide negative ions from ESI based on the addition of (6) Kebarle, P.; Tang, L. Anal. Chem. 1993,65, A!372-A986. (7)Tang, L.; Kebarle, P. Anal. Chem. 1991,63,2709-2715. (8) Tang, L.; Kebarle, P. Anal. Chem. 1993,65, 3654-3668. (9) Chowdhury, S. K.; Katta, V.; Chait, B. T.Biochem. Biophys. Res. Commun. 1990,167,686-692. (10) Perkins, J. R; Smith, B.; Gallagher, R T.;Jones, D. S.; Davis, S. C.; Hoffman, A. D.; Tomer, K B. J Am. SOC.Mass Spectrom. 1993,4, 670-684. (11) Chowdhury, S. IC; Katta,V.; Chait, B. T. J. Am. Chem. SOC.1990,112, 9012-9013. (12) Guevremont, R; Siu, K. W. M.; Leblanc, J. C. Y.; Berman, S. S. /. Am. SOC. Mass Spectrom. 1992,3, 216-224. (13) Feng, R; Konishi, Y. J. Am. SOC.Mass Spechom. 1993,4 , 638-645. (14) Loo, J. A; Loo, R R 0.;Udseth, H. R; Edmonds, C. G.; Smith, R D. Rapid Commun. Mass Spectrom. 1991,5, 101-105. (15) Loo, J. A; Udseth, H. R; Smith, R D. Rapid Commun. Mass Spectrom. 1990, 4,207. (16) Ashton, D.A; Reddell, C. R; Cooper, D. J.; Green, B. N.; Oliver, R W. A Org. M u s Spectrom. 1993,28, 721-728. (17) Leblanc, J. C. Y.; Beuchemin, D.; Siu, K W. M.; Guevremont, R; Berman, S.S. Ow. Mass Spectrom. 1991,26, 831-839. (18) Mirza, U. A; Cohen, S. L.; Chait, B. T. Anal. Chem. 1993,65,1-6.
0003-2700/95/0367-0586$9.00/0 Q 1995 American Chemical Society
acids to the oligonucleotide solution and the gas phase dissociation of oligonucleotide-diamine complexes in the atmospherevacuum interface region. EXPERIMENTAL SECTION Materials and Reagents. Oligonucleotides were purchased from Sigma (St. Louis, MO) and Pharmacia (piscataway, NJ) as sodium or ammonium salts. All the other reagents were from commercial sources and were used as received. Desalting of oligonucleotides was accomplished by exchanging with ammonium ions. The oligonucleotide samples were dissolved in 1.0 M ammonium acetate and concentrated using Centricon-3filters (Amicon, Beverly, MA). The process was repeated, and the oligonucleotidewas then dissolved in deionized water and treated with 5%(vlv) ammonium-saturatedAmberlite CG50 ion exchange resin (Sigma, St. Louis, MO) . The ammonium-saturated resin was prepared by treatment of Amberlite CG50 powder (purchased as proton form) with saturated ammonium acetate solution and subsequent washing with deionized water. The resin was removed from the oligonucleotide solution by centrifugation before analysis of the desalted oligonucleotide. The pH of oligonucleotide solutions was not measured directly due to the small volumes (typically 20-50 pL) used. The pH values were estimated using those measured for solutions of identical composition but without the oligonucleotide. Mass Spectrometer. A Sciex (Thornhill,ON, Canada) TAGA 6000E triple quadrupole mass spectrometer was used for this work. This mass spectrometer was equipped with a modified nozzle-skimmer electrospray interface, as described in previous publications.3Jg Countercurrent nitrogen flow was used to aid electrospray droplet desolvation, and coaxial Sk-6 sheath gas was used to suppress corona discharge. A sheathless ESI source optimized for the analysis of very small volumes of aqueous solutions at low infusion rates (0.2 pL/min) was used.20 A potential of -3.0 to -3.5 kV was used to produce a stable negative ESI current. The skimmer voltage was set to -65 V for all experiments, and the nozzle voltage was adjusted to achieve optimal desolvation and signal intensity of analyte ions. Unless otherwise indicated, the typical nozzle voltage used was -120 to -150 V. Procedures. In a typical ESI experiment, solutions of 0.15 mg/mL (ca. 40 pM for a 12-mer) of oligonucleotides in deionized water was directly infused into the ESI source using a syringe pump (Harvard, South Natick, MA). For charge state reduction experiments, the oligonucleotide was mixed with the solution containing the charge state reduction reagent in deionized water. For acidic reagents, the solution was mixed immediately before infusion to prevent possible decomposition of oligonucleotides. The oligonucleotide-acid mixtures were found to be stable for hours to days depending on the nature and the amounts of the acids present. RESULTS
Charge State Reduction by Addition of Acids to the Oligonucleotide Solution. The ESI mass spectrum of the sodium salt of oligonucleotide d(pT)lz in deionized water (pH
Ms+3Na
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1
I
C
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.
400
.
.
600
.
1,I
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.
800
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-
--
Figure I.Electrospray ionization mass spectra of d(pT)rz Na salt in (A) deionized water (pH 7.0); (8)0.05 M HOAc (pH 3.0); (C) 5 M HOAc (pH 1.9); and (D) 5 M HOAd0.3 M HCI (pH 0.5).
-
7.0) showed a charge state distribution with a maximum at 9F i e M). The extensive adduction of sodium cations is evident from the spectrum. It has recently been demonstrated that exchange with ammonium ions substantially reduces cation adduction in matrix-assisted laser desorption (MALDI) and ESI mass spectrometry experiments.21*22We also observed the efficient removal of metal cations from ESI solution upon cation exchange with ammonium ions, as illustrated in Figure 2A for d(pT)rz. M e r desalting, the sodium adduction was greatly reduced, allowing a precise molecular weight determination (calculated M, = 3668.4, observed M, = 3668.7 f 0.4). At the same time, the center of the charge state distribution shifted to higher m/z values (compare Figures l . 4 and 2A). This was attributed to the shielding of charge sites on the oligonucleotide by ammonium ions (vide infra). Direct addition of acids to the oligonucleotidesolution shifted the charge state distribution even more. As shown in Figures 1and 2, the addition of progressively higher proportions of acetic acid and formic acid shifted the center of the charge state distribution of d(pT)lz to progressively lower value (higher m/z) without appreciably affecting the sensitivity. Formic acid was observed to be more effective for charge state reduction than acetic acid, as expected from the difference in
N
(19) Smith,R D.;Loo, J. A; Barinaga, C. J.; Edmonds, C. G.; Udseth,H. R J Am. SOC.Mass Spectrom. 1990, 1,53-65.
(20)Gale, D.C.;Smith,R D.Rapid Commun. Mass Spectrom. 1993, 7,10171021.
(21) Nordhoff, E.; Ingendoh, A; Cramer, R; Overberg, A; Stahl, B.; Karas, M.; Hillenkamp, F.; Crain, P. F. Rapid Commun. Mass Spectrom. 1992,6,771776. (22) Stults, J. T.; Marsters, J. C. Rapid Commun. Mass Spectrom. 1991,5359363.
Analytical Chemistry, Vol. 67, No. 3, February 1, 1995
587
100
I M7-
A
'!
sc
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50
A
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AcO(Ac0H)
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.
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.
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.
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-
Figure 2. Mass spectra of d(pT)12 ammonium salt in (A) deionized water (pH 7.0); (B) 0.5 M HC02H (pH 1.9); (C) 2.5 M HC02H (pH 1.6); and (D) 2.5 M HC02HhO mM 1,lO-phenanthroline (pH * 1.7).
solution pKa (3.75 for formic acid vs 4.75 for acetic acid23). When 2.5 M formic acid was used, only 4- and 3- charge states remained detectable within the range of the mass spectrometer (m/z 1400) (Figure 2C and D). We have also evaluated the addition of stronger acids such as HC1, CF~COZH, and H3P04 with variable concentrations. While HC1 caused substantial reduction of signal accompanyingthe charge state shifts,CF3C02H and Hr PO4 also introduced peaks due to extensive clustering (to themselves and to oligonucleotides). The reduction of signal intensitywas apparent even when the concentrationsof these acids were reduced to keep all the charge states withii the m/z range of the mass spectrometer. Figure 3 compares the spectra when acetic acid and CF~COZHwere added to the oligonucleotide solution. The extensive clustering of CF3C02H can be identified readily. The addition of H3P04 produced even more extensive clustering (data not shown). We have also observed more intense signals in the low m/z region of the spectra when strong acids were used. The suppression of oligonucleotide ion signal from the addition of strong acids may be due to several different reasons. The major factor is likely the competition of anions from the strong acids (Cl-, CFsC02-, and all forms of phosphate anions) and their clusters (more important for CF~COZH and HsP03 for ionization. Since ESI generates a roughly constant ion current, an increase in the intensities of ions from added acids will reduce the intensities of the oligonucleotide ions.6-8 A second factor is the change in the ionization efficiency of the olgonucleotideswhen (23) CRC Handbook of Chemisty and Physics;Lide, D. R, Ed.;CRC Press: Boston, 1990 Vol. 71.
580 Analytical Chemistry, Vol. 67,No. 3,February 7, 1995
m/Z Figure 3. Mass spectra of d(pT)iZ ammonium salt in (A) 0.5 M HOAc (pH 2.4) and (B) 0.1 M CF3C02H (pH 0.9).
-
-
the solvent composition is ~hanged.~-*32~*2~ Oligonucleotides are very polar compounds and apparently require high desolvation energies to form gas phase ions. This is likely one of the major reasons for the difficulty in ionizing oligonucleotides using conventional techniques such as fast atom bombardment (FAJ3). The addition of weak organic acids like CH3C02H and HCOzH is expected to decrease the solvation energy and facilitate the ionization of oligonucleotides. A recent report has shown that addition of organic solvents substantially increased the signal intensity of a &mer oligonucleotide.26On the other hand, strong "inorganic" acids such as HC1, CF~COZH, and H3P04would not be expected to have such beneficial effects. In this work, we encountered more difficulty in maintaining a stable ESI current when using strong acids, possibly due to the higher conductance of the strongly acidic solutions. We conclude that strong inorganic acids are less suited for the charge state reduction of oligonucleotides. Interestingly, oligonucleotides in a mixture of HCl/CHr COzH yielded results as good as that from addition of HCOzH (Figures 1D and 2C). This again indicates the importance of solution composition in determining the extent of charge state reduction. The acid combination provides both acidity (PH 0.5, Figure 1D) and an organic environment, yielding better results from the mixture than from the individual components. We have also investigated the additions of organic solvents such as methanol and acetonitrile to oligonucleotide solutions in the presence of strong acids and observed some improvement in oligonucleotide ion intensity and ESI current stability, similar to the effect of acetic acid. A side effect of adding acids, especially strong acids, is that adducts of Fe(II) ions were introduced (Figures 1 and 2B and (24) Sakairi, M.; Yergey, A. L.; Siu, K W. M.; Leblanc, J. C. Y.; Guevremont, R; Berman, S. S. Anal. Chem. 1991,63, 1488-1490. (25) Hirabayashi, A; Takada, Y.; Kambara, H.; Umemura, Y.;Ito, H.; Kuchitsu, IC Chem. Php. Lett. 1993,204, 152-156. (26) Bleicher, K; Bayer, E. Biol. Mass Spectrom. 1994,23, 320-322.
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Figure 4. Mass spectra of d(pT)lZ Na salt in 10 mM butanediammonium diacetate (BDA) (pH 7.8) with AVNSof (A) -70; (8) -120; (C)-170; and (D)-220 V.
C), the identity of which was confirmed by high-resolution mass spectrometry measurements. The likely source of the FeOI) ions is the stainless steel syringe needle, used in the ESI source, through a combination of acid corrosion and electrospray processes (the correspondence of the electrospray process to an electrolytic cell has been discussed in depthnpB >. We have investigated the removal of the F e O adducts through the addition to oligonucleotide solution of compounds that will form stable chelating complexes with FeQI) and that will not form negative ions themselves. Crown ethers, cryptands, and many nitrogencontaining macrocyclic compounds all show some effect in removing FeOD adducts. However, some of these additives and their complexes form adducts with oligonucleotide ions and thus are not useful for the FeQI) removal. The most effective compound identified to date is l,l@phenanthroline. As shown in Figure 2D, addition of 50 mM l,l@phenanthroline to the oligonucleotide/acid solution removed FeQI) adducts quantitatively, without forming any new adducts or any interfering ions. Charge State Reduction through the Dissociation of Oligonucleotide-Diamine Complexes in the Interface Region. As shown by comparison of Figures lA and 4A, when 1,4butanediammonium diacetate is added to solutions containing d(pP12, the ESI spectra showed a charge state shirting (to higher (27) Blades, A. T.; Ikonomou,M. G.; Kebarle, P. Anal. Chem. 1991,63,21092114. (28)Van Berkel, G. J.; Mchckey, S. A; Glish, G. L.Anal. Chem. 1992,64, 1586- 1593.
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Figure 5. Mass spectra of d(pT)12 Na salt with AVNS of -220 V in solution of (A) 10 mM butanediammonium diacetate (pH 7.8); (B) 2.5mM butanediammonium diacetate (pH 7.7); and (C)deionized water (pH 7.0).
-
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m/z). The higher charge states are free from adduction, while the lower charge states show variable extents of diaminehodium association. Considering that the diammonium diacetate solution (pH 7.8) is more basic than the original oligonucleotide (p& and ~K,zfor 1,4butanediamine are 10.1 and 9.5, respectivelyz3), the reduction in charge states cannot be due to the change in the solution pH, as observed for addition of acids. The most likely explanation seems to be the shielding of the chargebearing sites by the diammonium ions and the subsequent dissociation of the oligonucleotide-diamine complexes. This hypothesis was sup ported by subsequent experiments in which the voltage difference between the nozzle and the skimmer across the atmospherevacuum interface, AVNS,was increased to induce greater collisional activation of ions.29 As shown in Figure 4B-D, the progressive increase in A V Nshifted ~ the center of charge state distribution to progressively higher m/z along with a gradual decrease in the intensity of diamine complexes. The diamine concentration was also important for the charge state reduction. Higher concentrations of diamine produced more extensive adduction to oligonucleotides and required higher A& to dissociate the complexes, while at lower concentrations diamine was less effective in reducing the charge states of oligonucleotides. Additionally, we observed that at a given AVN~,a higher concentration of diamine suppresses the backbone dissociation of oligonucleotide ions, as shown in Figure 5. At a AVNSof -220 V, extensive dissociation N
~
~~
(29) Barinaga, C. J.; Edmonds, C. G.; Udseth, H. R; Smith, R Moss Spectrom. 1989,3, 160-164.
D.Rapid Commun.
Analytical Chemistry, Vol. 67, No. 3, February 1, 1995
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of oligonucleotide d(pT)lz was observed (Figure 5C) when d(pT)~z was electrosprayed from deionized water, producing w, and (hT) ions and their sodium adducts, as expected from results of several previous studies on the dissociation of oligonucleotide anions under low-energy collisional activation conditions.30-32 At a 2.5 mM concentration, butanediamine substantially reduced the backbone dissociation (Figure 5B), and at 10 mM no backbone dissociation products were observed (Figure 5A). The dramatic difference in dissociation behavior of oligonucleotides under different solution conditions is attributed to two factors. The first is that addition of diamines produced oligonucleotide ions of lower charge states which are more stable relative to highly charged states due to both a decreased contribution from Coulombic repulsion and their lower kinetic energy in the nozzle-skimmer region. The second, and probably the more important factor here, is the “cooling” effect of the dissociation of the noncovalent oligonucleotide-diamine complexes which precedes backbone dissociation. The dissociation of each complex is estimated to consume 10-30 kcal/mol (0.5-1.5 eV, the estimated binding energy of the diamine-oligonucleotide complex), which should be a considerable portion of the total available energy to drive dissociation. These experiments demonstrate that by proper adjustment of diamine concentration and the magnitude of AVNS, an optimum condition can be achieved in which the charge states can be greatly reduced without decomposition of oligonucleotide ions (Figure 4). We have investigated several other diamines, including propanediamine, tetramethylbutanediamine, and hexanediamine. In each case, efficient charge state reduction of oligonucleotides could be obtained as with butanediamine. Figure 6 shows the charge state reduction achieved by dissociation of complexes of d(pT)12and hexanediamine. These experiments also show that larger diamines form stronger complexes with the oligonucleotidesions and that higher AVNSis needed to dissociate the complex (compare the value of AVNS used to dissociate oligonucleotide-diamine complexes for the 3- charge state in Figures 4 and 6). Charge State Reduction of a Small Oligonucleotide. We have applied both methods of charge state reduction to a smaller oligonucleotide, d(AGCT). As shown in Figure 7, the use of HCOzH and diamine both shifted the charge state of this 4mer oligonucleotide from mostly doubly and triply charged states to the singly charged species with little loss in sensitivity. Charge State Reduction of a Mixture of Oligonucleotides. To test the effect of charge state reduction on the mixture components analysis, we analyzed a mixture of oligonucleotides containing equal concentration of d(p‘Q12, d(pC)12, d(pA)lo, and d(pG)lo. These samples were purchased as sodium salts, and the mixture was desalted by ammonium ation exchange, as described in the Experimental Section. Figure 8A demonstrates the complexity of the ESI mass spectrum for this mixture. Each component gives several charge states, and some of the charge states overlap (e.g., both d(pT)12 and d(pA)lo contribute to the peak at m/z 520). The relative weak intensity of pd(A)lo and especially of d(pC)12 and d(pG)lo may be partly due to the differences in ionization efficiencyz6and partly due to differential (30) McLuckey, S. A; Van Berkel, G. J.; Glish, G. L. J. Am. SOC.Mass Spectmm. 1992,3, 60-70. (31) McLuckey, S. A; Habibi-Goudarzi, S.J Am. Chem. SOC.1993,115,1208512095. (32) Little, D. P.; Chorush, R A; Spier, J. P.; Senko, M. W.; Kelleher, N. L; McLafferty, F. W. J. Am. Chem. SOC.1994, 116, 4893-4897.
590 Analytical Chemistry, Vol. 67, No. 3, February 1, 1995
I
M5-
I
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.
.
.
.
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.
.
.
.
,
D
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-
Figure 6. Mass spectra of d(pT)12 ammonium salt in 5 mM hexanediammonium diacetate (HDA) (pH 8.1) with AVNs of (A) -70; (B) -100; (C)-180; (D)-250; and (E) -350 V.
loss of oligonucleotides during Centricon-3ultrafiltration. Upon addition of formic acid, the charge state distributions for all the components moved to higher m/z. As generally observed, the cation adduction (mostly due to Fe(II) and adventitious sodium ions) becomes more extensive for lower charge state ions. When 50 mM 1,lCLphenanthroline was added, the cation adducts were efficiently removed, and the spectrum consists of only one charge state from each oligonucleotides with the exception of d(pT)lz, for which two charge states remain in the spectrum (Figure 8C). Thus, after charge state reduction, the mass spectral complexity for the mixture is substantially reduced, and peaks from all components are well separated and easily identiiled. DISCUSSION What Determines the Charge State Distribution in ESI and Whether Charge State Reduction Is Achievable? The extensive multiple charging phenomenon of large molecular species is effectively unique to ESI. Although doubly charged and even more highly charged ions can sometimes be observed in other ionization processes such as FAB/LSIMS and, probably more often, in MALDI experiments, the multiple charging from ESI is general and extensive. This multiple charging phenomenon in ESI is not due to the intrinsic stability of multiply charged ions in the gas phase, but rather to the fact that ions are generated, under the influence of strong electrostatic fields, from solutions and highly charged liquid droplets. The formation of multiply
T
A
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M3-
G d(pG)io
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1200
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,
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- -
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Figure 7. Mass spectra of d(AGCT) ammonium salt in (A) deionized water (pH 7.0); (B) 10 mM tetramethylbutanediammoniumdiacetate (pH 8.2) with AVNS of -240 V; and (C) 0.5 M HC02H (pH 2.0).
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charged ions also attests to the very soft nature of the ESI process. In the gas phase, the highly charged species are more reactive toward acid-base processes than those with lower charge states due to Coulombic and possible conformational effect^.^^^^^ This has been demonstrated by charge state reduction in reactions of multiply protonated proteins with neutral bases in the gas pha~e.35-~~ Charge state reduction is also observed when the voltage difference across the atmospheric pressure-vacuum interface is increased.15J6 An increase in the voltage difference will cause more energetic collisions, and in addition to accelerating desolvation, these collisions can also cause fragmentation and charge exchange for ions of higher charge states. More recently, several groups have demonstrated the reduction to very low charge states of multiply protonated proteins through ionmolecule reactions in the gas p h a ~ e . ~ O - ~ ~ (33) Rockwood, A L.;Busman, M.; Smith, R D. Inf. J Mass Specfrom. Ion Processes 1991,111, 103-129. (34) McLuckey, S.A;Glish, G. L.; Van Berkel, G. J. In Proceedings of 39th ASMS Conference on Mass Spectrometry and Allied Topics;Nashville, TN,1991; pp 901-902. (35) McLuckey, S.A;Van Berkel, G. J.; Glish, G. L./. Am. Chem. SOC.1990, 112, 5668-5670. (36) McLuckey, S.A;Glish, G. L;Van Berkel, G. J. Anal. Chem. 1991,63, 1971-1978. (37) Loo, R R 0.; Loo, J. A; Udseth, H. R; Fulton, J. L.; Smith, R D. Rapid Commun. Mass Spectrom. 1992,6, 159-165. (38) Ogorzalek Loo, R R; Smith, R D. J. Am. SOC.Moss Specfrom. 1994,5, 207-220. (39) Cheng, X;Bakhtiar, R; Van Orden, S.; Smith, R D. Anal. Chem. 1994, 66, 2084-2087. (40) Wmger, B. E.;Light-Wahl, K. J.; Loo, R R 0.; Udseth, H. R; Smith, R D. J. Am. SOC.Mass Spectrom. 1993,4, 536-545.
,
.
,
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,
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800
,
,
1000
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Figure 8. Mass spectra of a mixture of d(pT)12, d(pC)w, d(pA)io, and d(pG)lo ammonium salts in (A) deionized water (pH 7.0); ( 8 ) 2 M HC02H (pH 1.7); and (C) 2 M HC02H/50 mM 1,lOphenanthroline (pH 1.8).
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Noncovalent associations, such as structurally specific complexation and nonspecik analyte aggregation usually observed with some milder ESI-MS interface conditions, frequently produce ions with lower charge states (higher m/z) than those for isolated (nonassociated) ions.",45 Possible explanations are that the association reduces the number of charge-bearing sites on the surface of the analyte ions and that the association frequently requires the adoption of specific structures/conformationswhich are more compact than in nonassociated ions."*& Thus the charge state distribution finally detected will be determined by ion conformation, initial charging in solution, and energetics and thermochemistry governing the reactions after the droplet formation. These reactions include proton transfer (acid-base reaction in the gas phase), desolvation, dissociation of complexes, and ion fragmentation. These considerations and observations suggest that charge state reduction should be possible by manipulation of solution composition and interface conditions. Charge state reduction should be especially favorable under interface conditions that facilitate charge exchange. (41) Cassady, C.;Wronka, J.; Kruppa, G. H.; Laukien, F. H. Rapid Commun. Mass Specfrom. 1994,8, 394-400. (42) Gross, D.S.;Schnier, P. D.; Williams, E. R In Proceedings of 42nd ASMS Conference on Mass Spectromehy and Allied Topics; Chicago, II+ 1994; p 1056. (43) Ogorzalek Loo, R R; Wmger, B. E.; Smith, R D.]. Am. SOC. Mass Spectrom., in press. (44)Smith, R D.; Light-Wahl, K J.; Winger, B. E.; Loo, J. A 0%.Moss Specfrom. 1992,27, 811-821. (45) Smith, R D.;Light-Wahl, K. J. Biol. Mass Spectrom. 1993,22, 493-501.
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Effect of Adding Acids to the Oligonucleotide Solution. The charge state reduction effects observed upon addition of acids for the ESI of oligonucleotides can be interpreted on the basis of the solution acid-base chemistry and reactions in the interface region. Solution pH can affect charge state distributions, and previous work with proteins and peptides has demonstrated the correlation of charge state distribution with the solution acidbase chemistry.12 The results were explained on the basis of the convolution of factors involved in different stages of the ESI process. The studies in protein systems are complicated by the possible conformation changes induced by pH modification. For oligonucleotides studied in this work, changes in solution pH are not expected to induce major changes in conformation. Noncovalent associations of the oligonucleotides can also be excluded due to the low concentration and the short sequence of the oligonucleotides used.46 Thus, we expect simple dependence of charge state distribution on solution pH for oligonucleotideswhen other conditions are unchanged, as observed in this work. The acidic component in solution will also affect the charge state distribution through reactions in the interface region. The ions formed in the interface region will undergo many collisions with solvent molecules (and more with gases added for desolvation). The more acidic the solution, the more likely the acids will donate protons to oligonucleotide anions and the more likely the charge states of oligonucleotide anions will be reduced. It has been reported that an increase in the distance between the electrospray needle tip and the sampling electrode (aperture) decreases the relative intensity of anions that have higher proton afEnities, an observation attributed to the proton transfer reactions during the spray process.47 Several studies have also shown that addition of acidic or basic compounds to the atmosphere-vacuum interface region changed the charge state distribution of proteins in the direction predicted on the basis of consideration of such proton transfer reaction~.3~*~~ In our case, it is difficult to separate the effect of solution acid-base equilibrium from that due to proton transfer reactions between multiply charged ions and solvent/ cosolute in the interface region. We do observe a slightly enhanced effect of charge state reduction from added acids when AVNS is increased, similar to other reports of charge state reductions by increasing ATINS. The more energetic collisions may drive reactions that may otherwise be too slow to be observed due to endothermicity or kinetic barriers. Since a nozzleskimmer voltage gradient must be generally applied for ion desolvation and transmission, reactions in the interface region may contribute to the charge state reduction of oligonucleotides when acids are present. Other mechanisms such as dissociative proton transfer of complexes of oligonucleotide ions with neutral acids during the electrospray process may also affect the charge state of the oligonucleotide ions. This type of process is similar to the second charge state reduction method reported in this paper and (46) In the case of the fourcomponent mixture of oligonucleotides, it is possible that noncovalent duplex and quadruplex DNA exist under appropriate solution conditions with annealing. See: Light-wahl, K J.; Springer, D. L.; Winger, B. E.; Edmonds, C. G.; Camp, D. G.; Thrall, B. D.; Smith, R D.]. Am. Chem. SOC.1993,115,803-804. Goodlett, D. R; Camp, D. G.; Hardin, C. C.; Corregan, M.; Smith, R D. Biol. Mass Spectrom. 1993,22,181-183. Edmonds, C. G.; Cheng, X;Bakhtiar, R; Van Orden, S.; Smith, R D.; Schlegel, S. C.; Camp, D. G., I1 In Proceedings of 42ndASMS Conference on Mass Spectromety and Allied Topics; Chicago, IL,1994; p 912. (47) Hiraoka, K; Murata, K; Kudaka, I. Rapid Commun. Mass Spectrom. 1993, 7, 363-373. (48) Lin, B.; Sunner, J. In Proceedings of 41st ASMS Conference on Mass Spectromety and Allied Topics; San Francisco, CA 1993; pp 1058a-1058b.
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has also been invoked recently in explaining the formation of protonated proteins from solutions of very high P H . ~However, ~ this is likely a minor contribution to oligonucleotide charge state shifting in the case of acetic and formic acids due to the absence of complexes of these acids to oligonucleotide ions, even under gentle ESI interface conditions. Effects of Adding Diamines to Oligonucleotide Solutions. In aqueous solution, d(pT)B shows a charge state distributionwith maximum intensity at 9-. Addition of organic bases might be expected to increase the charge states (based on the simple acidbase considerations discussed above). In contrast, we observed reduction in charge states (compare Figures 4A and 6A with Figures lA and 2A). In explaining these observations, we note that phosphodiesters in oligonucleotides are very strong acids which are completely ionized at pH 1-2, and that the nucleobases are deprotonated above pH 0, 2.3-2.5, 3.7-3.9, and 4.3-4.5 for T, G, A, and C, re~pectively.~.~~ Addition of bases will not produce more oligonucleotide anions in solution but will likely help to stabilize these anions in the interface region. (However, a recent paper reported increased sensitivity with increased pH of the solution up to pH 11for ESI of oligonucleotides.26) It can be seen in Figures 4 and 6 that diamine adducts of variable number with the oligonucleotide anions are formed for lower charge states but not for higher charged states. We propose that the charge state reduction observed here is due to the shielding (neutralization) of chargebearing sites (phosphodiesters) by formation of diamine adducts in solution. As a class of polyanions, oligonucleotides form adducts with polycationic compounds readily in s o l ~ t i o n ~ ~ ~ ~ ~ and in biological systems.50 Some important examples are the tRNA-spermine interaction, DNMRNA-protamine interaction, and packing of nuclear DNA around histone proteins.50 Upon transfer to the gas phase, the adducts may dissociate, depending on the interface conditions, producing oligonucleotide ions with lower charge states with or without adduction of amines (Scheme 1). Similar effects of counterions on the charge state distribution of positively charged peptides and proteins have also been noted.% It is clear that through the dissociation of oligonucleotidediamine complexes, the efficiency of charge state reduction will depend on the nature of the adducts; too weak an adduct will not efficiently shield the charge, and too strong an adduct will be difficultto dissociatewithout breaking covalent bonds, a side effect to be avoided. Diamines investigated in this work offered satisfactory results for small oligonucleotides. Other potentially more promising strategies of charge state reduction being explored in this laboratory include gas phase acid-base reactions in an FITCR mass spectrometer and derivatization of oligonucleotides for the formation of very low charge states from ESI. The derivatization (either to mask the negative charge under negative ion mode or to introduce a permanent positive charge under positive ion mode) combined with solution pH adjustment may (49) Le Blanc, J. C. Y.; Wang, J.; Siu, K. M. W.; Guevremont, R In Proceedings of 42ndASMS Conference on Mass Spectromety and Allied Topics;Chicago, IL, 1994 p 417. (50) Saenger, W. Principles of Nucleic Acid Structure; Springer-Verlag: New York, 1984. (51) CRC Practical Handbook of Biochemisty & Molecular Biology; Fasman, G. D., Ed.; CRC Press: Boston, 1992. (52) Frydman, L;Rossomando, P. C.; Frydman, V.; Femandez, C. 0.;Frydman, B.; Samejima, K Proc. Natl. Acad. Sei. U.S.A. 1 9 9 2 , 89, 9186-9190. (53) Stewart, K D.; Gray, T. A]. Phys. Org. Chem. 1 9 9 2 , 5, 461-466. (54) Mirza, U. A; Chait, B. T. Anal. Chem. 1 9 9 4 , 66,2898-2904.
Scheme 1 5'
3'
Gas-Phase Dissociation R,N-(CHz),-NR*
5'
3'
Singly Charged Oligonucleotide Ion
be a general, effective strategy for charge state reduction of large oligonucleotides. CONCLUSIONS Development of efficient methods for charge state reduction is important for extending ESI-MS to the analysis of mixture components. The charge state distribution of ions from ESI can be shifted by manipulating the ESI solution composition and the interface conditions. We have investigated two methods for charge state reduction of oligonucleotide negative ions from ESI. The methods have been designed on the basis of the consideration of the mechanism of ESI, the reactivity of multiply charged ions in the gas phase, and other factors that influence the charge state distribution. The first method involved the addition of acids to the oligonucleotide solution, and the observed charge state reduction effects are likely due to the solution and interface region acid-base reactions. The efficiency of charge state reduction was found to depend on the pKa,the concentration, and the nature of the acids. Acetic and formic acids were observed to be better
reagents than HCl, CF~COZH, and H3P04. While the former group of acids reduced the charge states of oligonucleotides without greatly affecting the signal intensity, the latter group of acids was found to substantiallyreduce the ion intensity. This adverse effect can be alleviated by using mixtures of acids such as HCl/CHr COZH. The second method investigated involved the formation of oligonucleotides-diamine complexes followed by the dissociation of the complexes in the interface region. The diamines neutralize the negative charges of the phosphodiesters and also suppress backbone dissociation (under conditions where the noncovalent complex dissociates). This method has the advantage that the stability of oligonucleotidesis not affected, but it requires the optimization of both the concentration of diamines and the interface dissociation conditions. Both methods showed satisfactory results for charge state reduction of oligonucleotides up to 1Zmers. Substantial reduction in spectral complexity was also observed for a fourcomponent mixture of oligonucleotides upon charge state reduction. We are currently investigating the utility of these and other charge state reduction methods in the analysis of DNA sequencing mixtures. ACKNOWLEDQMENT The authors wish to thank Drs. Karen J. Light-Wahl and Charles G. Edmonds for instrumental assistance and helpful discussions and an anonymous reviewer for pointing out the possible effects of dissociation of oligonucleotide-organic acid complexes on the charge state shifting of oligonucleotide ions. This work was supported by the Human Genome Program, Office of Health and Environmental Research, the US.Department of Energy. Pacific Northwest Laboratory is operated by Battelle Memorial Institute for the U.S. Department of Energy, through Contract No. DEAC0676RLO 1830. Received for review July 5, 1994. Accepted November 2, 1994. AC9406672 e Abstract published in Advance ACS
Abstracts, December 1, 1994.
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