RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 6 , 159-165 (1992)
Protein Structural Effects in Gas Phase Ion/Molecule Reactions with Diethylamine Rachel R. Ogorzalek Loo, Joseph A. Loo, Harold R. Udseth, John L. Fulton and Richard D. Smith* Chemical Methods and Separations Group, Chemical Sciences Department, Pacific Northwest Laboratory, Richland, WA 99352, USA
The relationship between gas-phase protein structure and ionhnolecule reactivity is explored in comparisons between native and disulfide-reduced aprotinin, lysozyme, and albumin. Reactions are performed in the atmospheric-pressureinlet to a quadrupole mass spectrometer employing a novel capillary interface-reactor. In reactions with equal concentrations of diethylamine, multiply protonated molecules generated by electrospray ionization (ESI) of ‘native’ proteins shifted to lower charge states than did multiply protonated molecules from ESI of the disulfide-reduced counterparts, suggesting that the disulfide-reduced protein ions are less reactive than native protein ions of the same charge state. Differences in reactivity may arise from protonation of different amino acid residues and/or differences in the proximities of charge sites in the two molecules. These results suggest that the reactivity of multiply charged proteins can be significantly affected by their gas-phase structure.
Investigations of the structure and reactivity of gasphase biomolecules generated by electrospray ionization (ESI) are increasing rapidly. By analogy to solution-phase chemistry, the reactivity of gas-phase biomolecules might be expected to depend significantly upon structure. Although it is uncertain to what extent gas-phase properties can be extrapolated from those observed in solution, and vice versa, methods which elucidate structural aspects may have significant bioanalytical and bio-physical utility. To date, most of the insights pertaining to charge-site location and three dimensional structure of multiply charged molecules have arisen from examination of observed ESI chargestate distributions in the gas phase and appear to reflect known characteristics in solution.’-” For example, a correlation has been noted for peptides and proteins between the maximum extent of protonation observed in positive-ion mass spectra from ESI of acidic solutions and the number of basic residues (generally arginine, lysine, and histidine). 1-4 Glutamine3,’ and ornithine6 residues have recently been implicated as protonation sites, as well, and a similar correlation has been noted between the maximum extent of deprotonation in negative ion ESI and the number of acidic residues.’ More significantly, several studies have demonstrated a relationship between protein solution conformation and ESI charge-state distribution^.^-^*^-'^ In those cases, where conformational changes were induced by altering solvent composition, pH, or temperature, or by reducing cysteine-cysteine disulfide bonds, differences in the extent of charging between native and denatured proteins have been largely attributed to differences in the availability of ionizable residues. Clearly, it is difficult to separate the solution-phase contributions to the ESI mass spectrum from the gasphase contributions. The correlation of maximum charge with number of basic or acidic sites does not pinpoint the actual charge sites in the gas-phase ion, and the dramatic differences observed between ESI mass spectra of different solution conformers are usually uninformative as to whether the structural Author to whom correspondence should be addressed.
0951-4198/92/030159-07 $05.00 01992 by John Wiley & Sons, Ltd.
differences persist in the gas phase. The key to investigating gas-phase properties specifically is to perturb the analyte when it is in the gas phase, not when it is in solution. Two approaches towards this end are collisional dissociation7. 14-21 and ion/molecule reactions.22-26 The collision dissociation of intact, multiply charged proteins and large polypeptides has been studied as a means of obtaining sequence-related information and ’*, I’ to explore other facets of protein structure.’, The effect of cysteine-cysteine disulfide bonds on fragmentation was studied in ribonuclease A16 serum albumins,” and proinsulin. I’ The dramatic differences which were observed in the dissociation spectra of native and disulfide-reduced proteins possessing the same number of charges were attributed to differences in charge location and to the additional bond cleavage(s) required to generate and observe certain products from the native protein. Tandem mass spectra (MUMS) of multiply protonated molecules arising from ESI of two different solution conformers of bovine ubiquitin were compared,’ and collisional dissociation in the atmospheric-pressure/vacuum interface has been applied to myoglobin and papain.13 However, collisional dissociation studies of ions of the same charge state so far have not yielded significant differences attributable to non-covalent structural elements.’ Such an observation is not surprising, since the collisional heating induced by multiple low-energy collisions may lead to isomerization and loss of any distinctive structural elements prior to dissociation. This report explores the relationship between a protein’s three dimensional structure in solution and its gas-phase ion/molecule reactivity. Ion-trap mass spectrometry (ITMS) experiments have demonstrated that ion/molecule reactions can efficiently shift the charge states of multiply protonated proteins and product ions to lower charge, potentially addressing questions regardin ion structure stability and charge-site 10cation!~-~~An advantage of ion/molecule reactions over collisional dissociation for probing structural effects is that extensive internal excitation of the ions is not required, so the studies can be performed under conditions more likely to preserve higher-order struc93
13314,
Received 31 December 1991 Accepted 15 January I992
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GAS PHASE ION/MOLECULE REACTIONS WITH DIETHYLAMINE
ture. By employing a novel capillary interfaceheactor which we have recently d e s ~ r i b e d , ~ ~these - ~ ' useful reactions can be transferred to any mass spectrometer. Comparisons between ESI mass spectra for native and denatured (disulfide-reduced) proteins are made. Collisional dissociation studies have previously suggested differences in fragmentation between native and disulfide-reduced proteins. 16, 18- l9 The purpose of the present study is to investigate whether or not those structural differences in gas-phase protein ions lead to differences in reactivity. EXPERIMENTAL The electrospray ionization mass spectrometer and capillary interface/flow reactor employed in these studies have been described e l s e ~ h e r e . ~Briefly, ~ - ~ ' the interfacelreactor is similar to the capillary design of Chowdhury et but it is constructed in the shape of a 'Y', providing two inlets and one outlet. The reactor was fabricated from three 6 cm lengths of 0.16 cm O D , 0.10cm I D stainless-steel tubing, silver soldered to a stainless-steel post drilled with a Y-shaped channel. An ESI s o u r ~ e " ~was ~ - ~positioned ~ 0.5-1 cm from one inlet, while diethylamine (DEA), boiling point 56.3 "C at 1atm, was introduced as a mixture of the room temperature vapor and air, flowing through 'shut-off' and needle valves to the second inlet. Based on the flow characteristics of the needle valve and reactor assembly, the D E A flow could range from 0 to 1X lo-* g/s, depending on the needle-valve setting employed. These estimates assumed that room temperature air at atmospheric pressure and saturated with the amine was delivered to the needle valve. Unfortunately, due to deficiencies in the D E A delivery system, the air was not saturated with amine at the highest flow rates studied. Consequently, we do not have good estimates of the D E A flows at the highest needle-valve settings, although the estimates for the lower settings (DEA flows 0 to 3 X g/s) are reliable. For each needlevalve setting, charge shifts observed in the ESI mass spectra were monitored over time to ensure that the D E A partial pressure in the inlet/reactor had attained a stable value. This precaution was particularly important when changing from a high reactant flow, where the air was not saturated with D E A , to a lower flow, where it could be saturated or nearly saturated. Desolvation was promoted in the stainless-steel interface/reactor by electrically heating the ~ a p i l l a r y .Capillary ~ ~ , ~ ~ temperature was controlled by adjusting the current (0-20 A) applied across the ESI inlet arm and the outlet arm; the arm which delivered the reactant amine was not heated. Very little heat transfer occurred between heated and unheated portions of the capillary. Electrical heating also permits examination of the temperature dependences for the ion/molecule reactions. Additional desolvation was achieved by collisional dissociation in the capillary outlet-skimmer region at capillary biases from 0-500 V relative to the grounded skimmer. The single quadrupole mass spectrometer used was constructed from Extrel components (Pittsburgh, PA, USA) and utilizes an additional radiofrequency-only quadrupole filter for improved ion focusing. To reduce the large decrease in sensitivity at higher m / z generally experienced with quadrupole mass spectrometers
under normal tuning conditions (where resolution increases linearly with mlz), resolution was adjusted to be constant over the 400-2000 mlz range investigated. Relative peak intensities are thus expected to better represent actual abundances than would typically be the case; however, sensitivity at higher mlz was still reduced somewhat, due to the fact that the capillary outlet/skimmer (0s)bias was held constant, while the optimum 0s bias depends on charge state.I4 In principle, the 0s bias could be scanned along with mlz, but the optimum voltages for low charge-state detection could collisionally dissociate higher charge states. With the exception of aprotinin (Novo BioLabs, Danbury, CT, USA), all biochemical samples and diethylamine were obtained from Sigma Chemical Company (St Louis, MO, USA) and were used without further purification. Reduction of disulfide bonds in lysozyme and albumin was carried out overnight at 37 "C in distilled water with 1,4-dithiothreitol (DTT)." Aprotinin was reduced wih DTT in a 5 0 m ~ (NH4)HC03pH 8.2 buffer for 10 min at 100 "C. RESULTS AND DISCUSSION Our ion/molecule reaction technique differs from the quadrupole ion-trap approach of McLuckey et al.22-24 in that we use much higher neutral reactant gas pressures to drive the reactions to shorter timescales. To yield detectable products in our apparatus, reactions must proceed sufficiently during the approximately millisecond transit time of the ions through the capillary reactor, while the ITMS experiments employed trapping times of up to a second with reactant pressures of to 10-6Torr. The higher gas pressures in our reactor have allowed an examination of reactions with smaller rate constants than those studied in the quadrupole ion trap.28 In principle, longer reaction times are possible with both approaches; pressures up to a few hundred Torr can be employed with the capillary reactor. Thermal reaction conditions dictated by the capillary temperature should apply to the inlet/reactor work, as opposed to the uncertain conditions of the iontrap studies. Also, the temperature dependence of the reaction rates can be explored by varying the capillary reactor temperature, potentially providing insights into the nature of the reactions. Because solvent and air are also present in the capillary reactor, additional interactions are possible, complicating some measurements, but also providing opportunites to explore the role of solvation in the reaction. Under the conditions used in this study, reactions occur after droplet evaporation and ion desorption/formation are expected to be complete, although the extent of macro-ion solvation observed will depend upon the capillary temperature. The supersonic expansion which follows the interaction region makes some of the interpretation, particularly with regard to clustering processes, more difficult. These differences do not present problems for the present study, since reactivities are compared under identical instrumental conditions. In the quadrupole ion trap (i.e., ITMS) and ion cyclotron resonance (ICR) trap, isolation of a single charge-state enables its reaction rate to be measured. While our approach shows only the combined effect of reaction of all of the charge states, examination of the
L
161
GAS PHASE ION/MOLECULE REACTIONS WITH DIETHYLAMINE
n+
(n-2)+
(n+4)+
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(n-3)+
(n-S)t
I 2
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loo0
10
mlr
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Figure 1. Simulated ESI charge-state distributions for (a) denatured and (b) native proteins (without diethylamine). Simulated ESI charge-state distributions after proton transfer reaction with diethylamine for multiply protonated molecules formed from (c) denatured and (d) native protein solutions, assuming that there is no difference in reactivity between e q u d y charged ions formed from the two solutions and given the two assumptions discussed in the text.
shifts in the charge-state distributions is still informative, particularly under conditions inducing large shifts, where the decrease in reaction rate with decreasing charge state is quite dramatic. Let us first consider how the ESI charge distributions would behave if all structural differences are lost in the gas phase (i.e., there is no difference in reactivity between equally charged ions formed from solutions of native and denatured proteins). ESI of the two solutions without any amine reactant should yield different charge distribution^,^.^.^-'^ as shown schematically in Fig. l ( a , b). Next, if we assume (i) that the reaction rates are much faster for the highest charge states than for the lower charge states and (ii) that the [M + nH]"+ charge state reacts to form an [ M + ( n - l)H]("-')+ charge state with reactivity equal to that of the [M + ( n - l)H]("-')+ species formed originally by ESI, then the two reaction product charge-state distributions should be almost identical, as shown in Fig. l(c,d). However, if equally charged ions formed from solutions of native and denatured proteins differ in their reactivities, the distributions after reaction should be quite different. Assumption (i) results in the highest charge states (i.e., those initially produced from ESI of the denatured protein) reacting with the amine quickly enough to eliminate any differences arising purely from the differing charge-state distributions of the conformers prior to reaction. Support for this assumption is
provided by the ITMS rate m e a s u r e m e n t ~ , which ~~.~~ have established that cytochrome c proton-transfer reaction rates decrease with decreasing charge state; e.g., the [M 10H]'"+ rate constant for proton transfer to dimethylamine is approximately one tenth that of the [M 14H]14+ charge state, and the [M 9HI9+ rate constant is less than one-fortieth that of the [M 10HJ1"+state. All proteins that we have examined show decreasing reactivity with decreasing charge state. In the studies reported here, and in other related studies,28ESI mass spectra are monitored as the reactant gas flow is varied. A t very low amine flow rates we have observed that several of the highest charge states from ESI of the denatured proteins disappear nearly simultaneously. (The reaction rates do not differ sufficiently for us to observe differences in their rates of disappearance.) A t higher reactant flows, where only low charge-state ions appear in the spectrum, the remaining higher charge states disappear nearly stepwise as flow is increased (e.g., the [M+nH]"+ ion decreases in abundance followed by the [M+ ( n - l)H]("-')+ ion and then the [M+(n-2)H]("-2)+ ion. We have taken care to conduct our studies in a high reactant-flow regime where assumption (i) is most likely to be justified. The validity of assumption (ii) is also quite important to this study. If the ions produced by the proton transfer reactions are not as reactive as equally charged ions produced directly by ESI (e.g., if the sites charged
+
+ +
+
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GAS PHASE ION/MOLECULE REACTIONS WITH DIETHYLAMINE
by ESI reflect energetically the most stable sites in solution, but not in the gas phase), interpretation of the charge-distribution shifts would be more difficult. Should assumption (ii) be invalid, bimodal charge-state distributions may be observed after proton transfer reaction, at least under certain experimental conditions, reflecting the different reaction rates for equally charged ions. No bimodal charge-state distributions were observed under the variety of conditions employed in this study. Furthermore, as individual charge states were monitored while the D E A flow rate was varied, no evidence of behavior that would invalidate assumptions (i) and (ii) was observed. For example, a peak for a multiply charged ion was not observed to initially decrease and subsequently increase in abundance. Direct measurement of ion/molecule reaction rates in ICR and quadrupole ion traps may provide further tests of these assumptions. Reactivities of equally charged ions are compared in this study, and the more reactive systems are defined as
47.1
1
the ones which yield the distribution with the lowest charge states after reaction with diethylamine. Alternatively, one could choose to define the more reactive systems as the ones which show the largest charge shifts; e.g., a sample for which the highest charge state observed shifted from 20+ to 10+ (10 charge states) could be deemed more reactive by this second definition than one which shifted from 15 + to 10 + ( 5 charge states). However, the latter definition is not used here because it might not adequately distinguish the greater reactivity of a molecule with a larger number of charges from a molecule having greater reactivity arising from structural differences. Figure 2(a) illustrates the ESI mass spectrum of hen egg-white lysozyme, ( M , 14306) in 5 % glacial acetic acid water with a methanol sheath solvent. All of the spectra in Figs 2 and 3 were obtained with outletskimmer biases of + 350 V. The capillary interface/ reactor temperature was 175 "C. As discussed previo~sly,~,the high charge states have been attri-
+
4; 1, [M+8HI8'
[M+lOHfo+
b)
500 500
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mlz Figure 2. ESI mass spectra of hen egg-white lysozyme (M,14 306) in 5% glacial acetic acid water with capillary outlet/skimmer bias of 350 V and capillary temperature 175 "C. (a) Without diethylamine, (b) with diethylamine.
+
h
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ml z Figure 3. ESI mass spectra of disulfide-reduced hen egg-white lysozyme in 5% glacial acetic acid + water with capillary outletlskimmer bias of 350 V and capillary temperature 175 "C. Detector sensitivity is approximately half that used in Fig. 2 (a) Without diethylamine, (b) with diethylamine.
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buted to a denatured conformation; lysozyme is known to switch from a compact, folded conformation to a random coil state upon lowering the pH from 3.3 to 1 .6.36Adding diethylamine to the capillary interface/ reactor shifts the charge-state distribution to lower charge (higher mlz) as shown in Fig. 2(b), obtained at a D E A flow rate of approximately 3 X 10-4g/s. Reduction of lysozyme's four disulfide linkages enables further charging in ESI,3.4as shown in Fig. 3(a), also obtained with an inlet/reactor temperature of 175 "C. This sample was also prepared in 5% glacial acetic acid +water and electrosprayed with methanol as the sheath solvent. Addition of D E A shifts the charge-state distribution to lower charge (Fig. 3(b)), but not to charge states as low as for the disulfide-intact protein, at the same D E A flow rate. Reaction products were examined under a variety of conditions, but there was never any evidence of the charge-state shift for the disulfide-reduced species equalling or exceeding that of the non-reduced species. We hypothesize that the lower charge states of the reduced protein are not as reactive as their equally charged counterparts in the non-reduced protein. Low intensity peaks which also shift to lower charge upon additon of D E A are evident in the mlz 1000-1700 region of the native (i.e., non-reduced) protein. (See Fig. 2(b) inset.) The charge-state distribution corresponds to a molecular weight of -45 kDa, and may arise from the heterogeneous glycoprotein o ~ a l b u m i n . ~Commercial ' ovalbumin samples yielded ESI mass spectra with and without D E A that were similar to those obtained for the lysozyme impurity. It would have been difficult to identify the impurity protein from the normal ESI mass spectrum of the sample without an ancillary separation process. However, peaks for the ovalbumin impurity shifted less (in m l z ) than peaks for lysozyme, reducing mass spectral overlap between the two species. This example shows that ion/molecule reactions may also have practical applications in evaluating mixtures. Bovine albumin ( M , 66430) is a globular protein possessing 17 disulfide linkages. The ESI mass spectrum of the native protein is shown in Fig. 4, along with the spectrum obtained after reaction with approximately 3 x g/s DEA. Figure 5 shows the corresponding mass spectra for the protein with its 17 disulfide linkages reduced. As observed for lysozyme, the nonreduced protein spectrum shifts to lower charge states than does that for the reduced protein. The highest charge state observed in positive-ion ESI of reduced bovine albumin shifts from about 95 + before reaction to about 70 + after reaction, while for native albumin, the shift is from about 63+ before reaction to 44+ after reaction. Further experiments on bovine pancreatic trypsin inhibitor (aprotinin, M , 6512) showed similar behavior (data not shown). ESI of the 'native' protein in 5% acetic acid +water with a methanol sheath solvent and 175 "C inlet/reactor yielded charge states ranging from 7 to 4 + , maximizing in abundance at the [M + 6HI6+ ion. After reaction with D E A at a flow rate of approximately 3 X g/s, only the 4 + and 5 + charge states were observed in our apparatus ( m / z limit 2000), with the 4 + ion about 5 times as abundant as the 5 + ions. Reduction of the 3 cysteine-cysteine disulfide bonds yielded an ESI mass spectrum with charge states rang-
+
ing from 8 + to 4 + before D E A reaction (maximum intensity at 6 + to 7 + ) and 6 + to 4 + after reaction (maximum intensity at 5 +). Here also, reaction products were examined under a variety of conditions, but the charge-state shift in the disulfide-reduced species never equalled or exceeded that of the non-reduced species. It would be very exciting and potentially useful if the differences in reaction rate between the disulfidereduced and non-reduced proteins reflected the retention of significant portions of the solution conformation. However, the observed reaction rate differences could simply reflect the comparison between a molecule which is fully extended vs a molecule which, while also significantly extended, is still restrained by disulfide bonds at certain locations. On the basis of these studies, we can only conclude that the proton-transfer reactivities of equally charged reduced and nonreduced protein ions differ. From the viewpoint of protein collision cross-sections, the greater reactivity observed for individual charge states in the non-
+ 100
0
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7)
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8 0
.d
u
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.d
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mlz Figure 4. ESI-MS of native bovine albumin (M,66 430) (a) without and (b) with diethylamine. Capillary outletlskimmer bias 350 V. Capillary temperature 175 "C.The sample was dissolved in 5% glacial acetic acid+water and sprayed with a methanol sheath solvent.
GAS PHASE ION/MOLECULE REACTIONS WITH DIETHYLAMINE
164
exert more and more of an effect with increasing charge. It is also possible that the higher order structure of the ions may be altered by the high concentrations of amine reactant or by thermal processes during transit through the heated capillary inletheactor. Finally, since it is known that solution conformation can dramatically affect the pK,'s of the individual amino acid constituents of proteins:' it is possible that ion/ molecule chemistry could also probe higher order structural effects arising from weak (e.g., hydrogen bonding) interactions. Additional ESI investigations exploring these issues are being pursued. CONCLUSION A capillary interface/reactor has been used to study ion/molecule proton-transfer reactions of multiply protonated proteins generated by ESI. The disulfidereduced protein ions studied here appear to be substantially less reactive to proton transfer than 'native' protein ions with the same number of charges. In the case of proton-transfer reactions, it is expected that the increased Coulombic forces arising from different comformations and charge locations (i.e., specific charge sites and their proximities) should increase the rate and exothermicity of proton transfer from multiply charged proteins. Further studies aimed at understanding the differences in reactivity and the structure and chemistry of multiply charged macro-ions in the gas phase are in progress.
Acknowledgements
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We acknowledge W. H. Robins and C. G. Edmonds for helpful discussions, and the US Department of Energy, Office of Health and Environmental Research, through internal Exploratory Research of the Molecular Science Research Center (Contract DE-AC06-76RLO 1830) for support of this research. Pacific Northwest Laboratory is operated by Battelle Memorial Institute.
2000
mlz Figures. ESI-MS of reduced bovine albumin (a) without and (b) with diethylamine. Capillary outlet/skimmer bias 350 V. Capillary temperature 175 "C. The sample was dissolved in 5% glacial acetic acid water and sprayed with a methanol sheath solvent.
+
reduced proteins might be surprising, since the more extended structures expected for the disulfide-reduced proteins should result in larger collision cross-sections. However, the proton-transfer reactions relevant here occur at rates far below their collision rate.= When the Coulombic forces arising from charge repulsion are also considered, it seems likely that a highly charged, nonreduced protein would be more reactive than an equally charged reduced protein, because the charge states would most likely be spaced more closely in the more constrained conf~rmation.~' In such instances, the increased Coulombic forces should increase the rate and exothermicity of proton transfer from multiply charged proteins. There are several other considerations worthy of further investigation. We have considered protein conformation as two cases-native and denatured (i.e., reduced), but our analysis has not incorporated the possibility that the gas-phase conformation may vary substantially with charge state as Coulombic forces
REFERENCES 1. J. A. Loo, H. R. Udseth and R. D. Smith, Biomed. Environ. Mass Spectrom. 17, 411 (1988). 2. J. A. Loo, H. R. Udseth and R. D. Smith, Anal. Biochem. 179, 404 (1989). 3. J. A. Loo, C. G. Edmonds, H. R. Udseth and R. D. Smith, Anal. Chem. 62, 693 (1990). 4. R. D. Smith, J . A. Loo, C. G. Edmonds, C. J. Barinaga and H. R. Udseth, Anal. Chem. 62, 882 (1990). 5. W. T. Moore, M. J.-F. Suter, T. B. Farmer and R. M. Capnoli, Proceedings of the 39th Conference on Mass Spectrometry and Allied Topics, pp. 256-257, Nashville, TN, ASMS, East Lansing (1991). 6. M. Sakairi and A. L. Yergey, Rapid Commun. Mass Spectrom. 5 , 349 (1991). 7. J. A. Loo, R. R. Ogorzalek Loo, K. J. Light, C. G. Edmonds and R. D. Smith, Anal. Chem. (in press). 8. S. K. Chowdhury, V.,Katta and B. T. Chait, J . A m . Chem. SOC. 112, 9012 (1990). 9. J. A. Loo, R. R. Ogorzalek Loo, H. R. Udseth, C. G. Edmonds and R . D. Smith, Rapid Commun. Mass Spectrom. 5,101 (1991). 10. V. Katta and B. T. Chait, Rapid Commun. Mass Spectrom. 5 , 214 (1991). 11. J. C. Y. Le Blanc, D. Beuchemin, K. W. M. Siu, R. Guevremont and S. S. Berman, Org. Mass Spectrom. 26, 831 (1991). 12. V. Katta and B. T. Chait, J. A m . Chem. SOC.113, 8534 (1991). 13. R. Feng, Y. Konishi, Proceedings of the 39th Conference on Mass Spectrometry and Allied Topics, pp. 1432-1433, Nashville, TN, ASMS, East Lansing (1991). 14. R. D. Smith, J. A. Loo, C. J. Barinaga, C. G. Edmonds and H. R. Udseth, J . A m . SOC. Mass Spectrom. 1, 53 (1989).
GAS PHASE IONlMOLECULE REACTIONS WITH DIETHYLAMINE 15. J. A. Loo, H. R. Udseth and R. D. Smith, Anal. Biochem. 179, 404 (1989). 16. J. A. Loo, C. G. Edmonds and R. D. Smith, Science 248, 201 (1990). 17. J. A. Loo, C. G. Edmonds, H. R. Udseth and R. D. Smith, Anal. Chim. Acta 241, 167 (1990). 18. J. A. Loo, C. G. Edmonds and R. D. Smith, Anal. Chem. 63, 2488 (1991). 19. J. A. Loo, C. G. Edmonds, R. R. Ogorzalek Loo, H. R. Udseth and R. D. Smith, in Experimental Mass Spectrometry, ed. by D. H. Russell, Plenum Press, New York (in press). 20. R. Feng, F. Bouthillier, Y. Konishi and M. Cygler, Proceedings of the 39th Conference on Mass Spectrometry and Allied Topics, pp. 1159-1160, Nashville, TN, ASMS, East Lansing (1991). 21. J. A. Loo, J. P. Quinn, S. I. Ryu, K. D. Henry, M. W. Senko and F. W. McLafferty, Proc. Natl Acad. Sci. USA (submitted). 22. S. A. McLuckey, G. J . Van Berkel and G. L. Glish, Proceedings of the 38th Conference on Mass Spectrometry and Allied Topics, pp. 1134-1135, Tucson, AZ, ASMS, East Lansing (1990). 23. S. A. McLuckey, G. J. Van Berkel, G. L. Glish, J. A m . Chem. SOC.112, 5668 (1990). 24. S. A. McLuckey, G. L. Glish and G. J. Van Berkel, Anal. Chem. 63, 1971 (1991). 25. R. R. Ogorzalek Loo, H. R. Udseth and R. D. Smith, J . Phys. Chem. 95, 6412 (1991). 26. R. R. Ogorzalek Loo, H. R. Udseth and R. D. Smith, Proceedings of the 39th Conference on Mass Spectrometry and
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Allied Topics, pp. 266-267, Nashville, TN, ASMS, East Lansing (1991). 27. B. E. Winger, K. J. Light and R. D. Smith, J. A m . SOC.Mass Spectrom. (submitted). 28. R. R. Ogorzalek Loo, H. R. Udseth and R. D. Smith (in preparation). 29. S. K. Chowdhury, V. Katta and B. T. Chait, Rapid Commun. Mass Spectrom. 4, 81 (1990). 30. J. A. Loo, H. R. Udseth and R. D. Smith, Rapid Commun. Mass Spectrom. 2, 207 (1988). 31. C. J. Barinaga, C. G. Edmonds, H. R. Udseth and R. D. Smith, Rapid Commun. Mass Spectrom. 3, 16 (1989). 32. R. D. Smith, C. J. Barinaga and H. R. Udseth, Anal. Chem. 60, 1948 (1988). 33. A. L. Rockwood, M. Busman, H. R. Udseth and R. D. Smith, Rapid Commun. Mass Spectrom. 5 , 582 (1991). 34. M. Busman, A. L. Rockwood and R. D. Smith J. Phys. Chem. (in press). 35. W. W. Cleland, Biochemistry 3, 480 (1964). 36. C. C. McDonald, W. D. Phillips and J. D. Glickson, J . A m . Chem. SOC.93, 235 (1971). 37. J. Caslavska, P. Gebauer and W. Thormann, J. Chromatogr. 585, 145 (1991). 38. A. L. Rockwood, M. Busman and R. D. Smith, Int. J . Mass Spectrorn. Ion Processes (in press). 39. E. D. Anderson, W. J. Becktel, F. W. Dahlquist, Biochemistry 29, 2403 (1990).
Protein Structural Effects in Gas Phase Ion/Molecule Reactions with Diethylamine Rachel R. Ogorzalek Loo, Joseph A. Loo, Harold R. Udseth, John L. Fulton and Richard D. Smith* Chemical Methods and Separations Group, Chemical Sciences Department, Pacific Northwest Laboratory, Richland, WA 99352, USA
The relationship between gas-phase protein structure and ionhnolecule reactivity is explored in comparisons between native and disulfide-reduced aprotinin, lysozyme, and albumin. Reactions are performed in the atmospheric-pressureinlet to a quadrupole mass spectrometer employing a novel capillary interface-reactor. In reactions with equal concentrations of diethylamine, multiply protonated molecules generated by electrospray ionization (ESI) of ‘native’ proteins shifted to lower charge states than did multiply protonated molecules from ESI of the disulfide-reduced counterparts, suggesting that the disulfide-reduced protein ions are less reactive than native protein ions of the same charge state. Differences in reactivity may arise from protonation of different amino acid residues and/or differences in the proximities of charge sites in the two molecules. These results suggest that the reactivity of multiply charged proteins can be significantly affected by their gas-phase structure.
Investigations of the structure and reactivity of gasphase biomolecules generated by electrospray ionization (ESI) are increasing rapidly. By analogy to solution-phase chemistry, the reactivity of gas-phase biomolecules might be expected to depend significantly upon structure. Although it is uncertain to what extent gas-phase properties can be extrapolated from those observed in solution, and vice versa, methods which elucidate structural aspects may have significant bioanalytical and bio-physical utility. To date, most of the insights pertaining to charge-site location and three dimensional structure of multiply charged molecules have arisen from examination of observed ESI chargestate distributions in the gas phase and appear to reflect known characteristics in solution.’-” For example, a correlation has been noted for peptides and proteins between the maximum extent of protonation observed in positive-ion mass spectra from ESI of acidic solutions and the number of basic residues (generally arginine, lysine, and histidine). 1-4 Glutamine3,’ and ornithine6 residues have recently been implicated as protonation sites, as well, and a similar correlation has been noted between the maximum extent of deprotonation in negative ion ESI and the number of acidic residues.’ More significantly, several studies have demonstrated a relationship between protein solution conformation and ESI charge-state distribution^.^-^*^-'^ In those cases, where conformational changes were induced by altering solvent composition, pH, or temperature, or by reducing cysteine-cysteine disulfide bonds, differences in the extent of charging between native and denatured proteins have been largely attributed to differences in the availability of ionizable residues. Clearly, it is difficult to separate the solution-phase contributions to the ESI mass spectrum from the gasphase contributions. The correlation of maximum charge with number of basic or acidic sites does not pinpoint the actual charge sites in the gas-phase ion, and the dramatic differences observed between ESI mass spectra of different solution conformers are usually uninformative as to whether the structural Author to whom correspondence should be addressed.
0951-4198/92/030159-07 $05.00 01992 by John Wiley & Sons, Ltd.
differences persist in the gas phase. The key to investigating gas-phase properties specifically is to perturb the analyte when it is in the gas phase, not when it is in solution. Two approaches towards this end are collisional dissociation7. 14-21 and ion/molecule reactions.22-26 The collision dissociation of intact, multiply charged proteins and large polypeptides has been studied as a means of obtaining sequence-related information and ’*, I’ to explore other facets of protein structure.’, The effect of cysteine-cysteine disulfide bonds on fragmentation was studied in ribonuclease A16 serum albumins,” and proinsulin. I’ The dramatic differences which were observed in the dissociation spectra of native and disulfide-reduced proteins possessing the same number of charges were attributed to differences in charge location and to the additional bond cleavage(s) required to generate and observe certain products from the native protein. Tandem mass spectra (MUMS) of multiply protonated molecules arising from ESI of two different solution conformers of bovine ubiquitin were compared,’ and collisional dissociation in the atmospheric-pressure/vacuum interface has been applied to myoglobin and papain.13 However, collisional dissociation studies of ions of the same charge state so far have not yielded significant differences attributable to non-covalent structural elements.’ Such an observation is not surprising, since the collisional heating induced by multiple low-energy collisions may lead to isomerization and loss of any distinctive structural elements prior to dissociation. This report explores the relationship between a protein’s three dimensional structure in solution and its gas-phase ion/molecule reactivity. Ion-trap mass spectrometry (ITMS) experiments have demonstrated that ion/molecule reactions can efficiently shift the charge states of multiply protonated proteins and product ions to lower charge, potentially addressing questions regardin ion structure stability and charge-site 10cation!~-~~An advantage of ion/molecule reactions over collisional dissociation for probing structural effects is that extensive internal excitation of the ions is not required, so the studies can be performed under conditions more likely to preserve higher-order struc93
13314,
Received 31 December 1991 Accepted 15 January I992
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GAS PHASE ION/MOLECULE REACTIONS WITH DIETHYLAMINE
ture. By employing a novel capillary interfaceheactor which we have recently d e s ~ r i b e d , ~ ~these - ~ ' useful reactions can be transferred to any mass spectrometer. Comparisons between ESI mass spectra for native and denatured (disulfide-reduced) proteins are made. Collisional dissociation studies have previously suggested differences in fragmentation between native and disulfide-reduced proteins. 16, 18- l9 The purpose of the present study is to investigate whether or not those structural differences in gas-phase protein ions lead to differences in reactivity. EXPERIMENTAL The electrospray ionization mass spectrometer and capillary interface/flow reactor employed in these studies have been described e l s e ~ h e r e . ~Briefly, ~ - ~ ' the interfacelreactor is similar to the capillary design of Chowdhury et but it is constructed in the shape of a 'Y', providing two inlets and one outlet. The reactor was fabricated from three 6 cm lengths of 0.16 cm O D , 0.10cm I D stainless-steel tubing, silver soldered to a stainless-steel post drilled with a Y-shaped channel. An ESI s o u r ~ e " ~was ~ - ~positioned ~ 0.5-1 cm from one inlet, while diethylamine (DEA), boiling point 56.3 "C at 1atm, was introduced as a mixture of the room temperature vapor and air, flowing through 'shut-off' and needle valves to the second inlet. Based on the flow characteristics of the needle valve and reactor assembly, the D E A flow could range from 0 to 1X lo-* g/s, depending on the needle-valve setting employed. These estimates assumed that room temperature air at atmospheric pressure and saturated with the amine was delivered to the needle valve. Unfortunately, due to deficiencies in the D E A delivery system, the air was not saturated with amine at the highest flow rates studied. Consequently, we do not have good estimates of the D E A flows at the highest needle-valve settings, although the estimates for the lower settings (DEA flows 0 to 3 X g/s) are reliable. For each needlevalve setting, charge shifts observed in the ESI mass spectra were monitored over time to ensure that the D E A partial pressure in the inlet/reactor had attained a stable value. This precaution was particularly important when changing from a high reactant flow, where the air was not saturated with D E A , to a lower flow, where it could be saturated or nearly saturated. Desolvation was promoted in the stainless-steel interface/reactor by electrically heating the ~ a p i l l a r y .Capillary ~ ~ , ~ ~ temperature was controlled by adjusting the current (0-20 A) applied across the ESI inlet arm and the outlet arm; the arm which delivered the reactant amine was not heated. Very little heat transfer occurred between heated and unheated portions of the capillary. Electrical heating also permits examination of the temperature dependences for the ion/molecule reactions. Additional desolvation was achieved by collisional dissociation in the capillary outlet-skimmer region at capillary biases from 0-500 V relative to the grounded skimmer. The single quadrupole mass spectrometer used was constructed from Extrel components (Pittsburgh, PA, USA) and utilizes an additional radiofrequency-only quadrupole filter for improved ion focusing. To reduce the large decrease in sensitivity at higher m / z generally experienced with quadrupole mass spectrometers
under normal tuning conditions (where resolution increases linearly with mlz), resolution was adjusted to be constant over the 400-2000 mlz range investigated. Relative peak intensities are thus expected to better represent actual abundances than would typically be the case; however, sensitivity at higher mlz was still reduced somewhat, due to the fact that the capillary outlet/skimmer (0s)bias was held constant, while the optimum 0s bias depends on charge state.I4 In principle, the 0s bias could be scanned along with mlz, but the optimum voltages for low charge-state detection could collisionally dissociate higher charge states. With the exception of aprotinin (Novo BioLabs, Danbury, CT, USA), all biochemical samples and diethylamine were obtained from Sigma Chemical Company (St Louis, MO, USA) and were used without further purification. Reduction of disulfide bonds in lysozyme and albumin was carried out overnight at 37 "C in distilled water with 1,4-dithiothreitol (DTT)." Aprotinin was reduced wih DTT in a 5 0 m ~ (NH4)HC03pH 8.2 buffer for 10 min at 100 "C. RESULTS AND DISCUSSION Our ion/molecule reaction technique differs from the quadrupole ion-trap approach of McLuckey et al.22-24 in that we use much higher neutral reactant gas pressures to drive the reactions to shorter timescales. To yield detectable products in our apparatus, reactions must proceed sufficiently during the approximately millisecond transit time of the ions through the capillary reactor, while the ITMS experiments employed trapping times of up to a second with reactant pressures of to 10-6Torr. The higher gas pressures in our reactor have allowed an examination of reactions with smaller rate constants than those studied in the quadrupole ion trap.28 In principle, longer reaction times are possible with both approaches; pressures up to a few hundred Torr can be employed with the capillary reactor. Thermal reaction conditions dictated by the capillary temperature should apply to the inlet/reactor work, as opposed to the uncertain conditions of the iontrap studies. Also, the temperature dependence of the reaction rates can be explored by varying the capillary reactor temperature, potentially providing insights into the nature of the reactions. Because solvent and air are also present in the capillary reactor, additional interactions are possible, complicating some measurements, but also providing opportunites to explore the role of solvation in the reaction. Under the conditions used in this study, reactions occur after droplet evaporation and ion desorption/formation are expected to be complete, although the extent of macro-ion solvation observed will depend upon the capillary temperature. The supersonic expansion which follows the interaction region makes some of the interpretation, particularly with regard to clustering processes, more difficult. These differences do not present problems for the present study, since reactivities are compared under identical instrumental conditions. In the quadrupole ion trap (i.e., ITMS) and ion cyclotron resonance (ICR) trap, isolation of a single charge-state enables its reaction rate to be measured. While our approach shows only the combined effect of reaction of all of the charge states, examination of the
L
161
GAS PHASE ION/MOLECULE REACTIONS WITH DIETHYLAMINE
n+
(n-2)+
(n+4)+
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1
(n-3)+
(n-S)t
I 2
500
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500
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loo0
10
mlr
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Figure 1. Simulated ESI charge-state distributions for (a) denatured and (b) native proteins (without diethylamine). Simulated ESI charge-state distributions after proton transfer reaction with diethylamine for multiply protonated molecules formed from (c) denatured and (d) native protein solutions, assuming that there is no difference in reactivity between e q u d y charged ions formed from the two solutions and given the two assumptions discussed in the text.
shifts in the charge-state distributions is still informative, particularly under conditions inducing large shifts, where the decrease in reaction rate with decreasing charge state is quite dramatic. Let us first consider how the ESI charge distributions would behave if all structural differences are lost in the gas phase (i.e., there is no difference in reactivity between equally charged ions formed from solutions of native and denatured proteins). ESI of the two solutions without any amine reactant should yield different charge distribution^,^.^.^-'^ as shown schematically in Fig. l ( a , b). Next, if we assume (i) that the reaction rates are much faster for the highest charge states than for the lower charge states and (ii) that the [M + nH]"+ charge state reacts to form an [ M + ( n - l)H]("-')+ charge state with reactivity equal to that of the [M + ( n - l)H]("-')+ species formed originally by ESI, then the two reaction product charge-state distributions should be almost identical, as shown in Fig. l(c,d). However, if equally charged ions formed from solutions of native and denatured proteins differ in their reactivities, the distributions after reaction should be quite different. Assumption (i) results in the highest charge states (i.e., those initially produced from ESI of the denatured protein) reacting with the amine quickly enough to eliminate any differences arising purely from the differing charge-state distributions of the conformers prior to reaction. Support for this assumption is
provided by the ITMS rate m e a s u r e m e n t ~ , which ~~.~~ have established that cytochrome c proton-transfer reaction rates decrease with decreasing charge state; e.g., the [M 10H]'"+ rate constant for proton transfer to dimethylamine is approximately one tenth that of the [M 14H]14+ charge state, and the [M 9HI9+ rate constant is less than one-fortieth that of the [M 10HJ1"+state. All proteins that we have examined show decreasing reactivity with decreasing charge state. In the studies reported here, and in other related studies,28ESI mass spectra are monitored as the reactant gas flow is varied. A t very low amine flow rates we have observed that several of the highest charge states from ESI of the denatured proteins disappear nearly simultaneously. (The reaction rates do not differ sufficiently for us to observe differences in their rates of disappearance.) A t higher reactant flows, where only low charge-state ions appear in the spectrum, the remaining higher charge states disappear nearly stepwise as flow is increased (e.g., the [M+nH]"+ ion decreases in abundance followed by the [M+ ( n - l)H]("-')+ ion and then the [M+(n-2)H]("-2)+ ion. We have taken care to conduct our studies in a high reactant-flow regime where assumption (i) is most likely to be justified. The validity of assumption (ii) is also quite important to this study. If the ions produced by the proton transfer reactions are not as reactive as equally charged ions produced directly by ESI (e.g., if the sites charged
+
+ +
+
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GAS PHASE ION/MOLECULE REACTIONS WITH DIETHYLAMINE
by ESI reflect energetically the most stable sites in solution, but not in the gas phase), interpretation of the charge-distribution shifts would be more difficult. Should assumption (ii) be invalid, bimodal charge-state distributions may be observed after proton transfer reaction, at least under certain experimental conditions, reflecting the different reaction rates for equally charged ions. No bimodal charge-state distributions were observed under the variety of conditions employed in this study. Furthermore, as individual charge states were monitored while the D E A flow rate was varied, no evidence of behavior that would invalidate assumptions (i) and (ii) was observed. For example, a peak for a multiply charged ion was not observed to initially decrease and subsequently increase in abundance. Direct measurement of ion/molecule reaction rates in ICR and quadrupole ion traps may provide further tests of these assumptions. Reactivities of equally charged ions are compared in this study, and the more reactive systems are defined as
47.1
1
the ones which yield the distribution with the lowest charge states after reaction with diethylamine. Alternatively, one could choose to define the more reactive systems as the ones which show the largest charge shifts; e.g., a sample for which the highest charge state observed shifted from 20+ to 10+ (10 charge states) could be deemed more reactive by this second definition than one which shifted from 15 + to 10 + ( 5 charge states). However, the latter definition is not used here because it might not adequately distinguish the greater reactivity of a molecule with a larger number of charges from a molecule having greater reactivity arising from structural differences. Figure 2(a) illustrates the ESI mass spectrum of hen egg-white lysozyme, ( M , 14306) in 5 % glacial acetic acid water with a methanol sheath solvent. All of the spectra in Figs 2 and 3 were obtained with outletskimmer biases of + 350 V. The capillary interface/ reactor temperature was 175 "C. As discussed previo~sly,~,the high charge states have been attri-
+
4; 1, [M+8HI8'
[M+lOHfo+
b)
500 500
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2000
mlz Figure 2. ESI mass spectra of hen egg-white lysozyme (M,14 306) in 5% glacial acetic acid water with capillary outlet/skimmer bias of 350 V and capillary temperature 175 "C. (a) Without diethylamine, (b) with diethylamine.
+
h
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ml z Figure 3. ESI mass spectra of disulfide-reduced hen egg-white lysozyme in 5% glacial acetic acid + water with capillary outletlskimmer bias of 350 V and capillary temperature 175 "C. Detector sensitivity is approximately half that used in Fig. 2 (a) Without diethylamine, (b) with diethylamine.
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GAS PHASE ION/MOLECULE REACTIONS WITH DIETHYLAMINE
buted to a denatured conformation; lysozyme is known to switch from a compact, folded conformation to a random coil state upon lowering the pH from 3.3 to 1 .6.36Adding diethylamine to the capillary interface/ reactor shifts the charge-state distribution to lower charge (higher mlz) as shown in Fig. 2(b), obtained at a D E A flow rate of approximately 3 X 10-4g/s. Reduction of lysozyme's four disulfide linkages enables further charging in ESI,3.4as shown in Fig. 3(a), also obtained with an inlet/reactor temperature of 175 "C. This sample was also prepared in 5% glacial acetic acid +water and electrosprayed with methanol as the sheath solvent. Addition of D E A shifts the charge-state distribution to lower charge (Fig. 3(b)), but not to charge states as low as for the disulfide-intact protein, at the same D E A flow rate. Reaction products were examined under a variety of conditions, but there was never any evidence of the charge-state shift for the disulfide-reduced species equalling or exceeding that of the non-reduced species. We hypothesize that the lower charge states of the reduced protein are not as reactive as their equally charged counterparts in the non-reduced protein. Low intensity peaks which also shift to lower charge upon additon of D E A are evident in the mlz 1000-1700 region of the native (i.e., non-reduced) protein. (See Fig. 2(b) inset.) The charge-state distribution corresponds to a molecular weight of -45 kDa, and may arise from the heterogeneous glycoprotein o ~ a l b u m i n . ~Commercial ' ovalbumin samples yielded ESI mass spectra with and without D E A that were similar to those obtained for the lysozyme impurity. It would have been difficult to identify the impurity protein from the normal ESI mass spectrum of the sample without an ancillary separation process. However, peaks for the ovalbumin impurity shifted less (in m l z ) than peaks for lysozyme, reducing mass spectral overlap between the two species. This example shows that ion/molecule reactions may also have practical applications in evaluating mixtures. Bovine albumin ( M , 66430) is a globular protein possessing 17 disulfide linkages. The ESI mass spectrum of the native protein is shown in Fig. 4, along with the spectrum obtained after reaction with approximately 3 x g/s DEA. Figure 5 shows the corresponding mass spectra for the protein with its 17 disulfide linkages reduced. As observed for lysozyme, the nonreduced protein spectrum shifts to lower charge states than does that for the reduced protein. The highest charge state observed in positive-ion ESI of reduced bovine albumin shifts from about 95 + before reaction to about 70 + after reaction, while for native albumin, the shift is from about 63+ before reaction to 44+ after reaction. Further experiments on bovine pancreatic trypsin inhibitor (aprotinin, M , 6512) showed similar behavior (data not shown). ESI of the 'native' protein in 5% acetic acid +water with a methanol sheath solvent and 175 "C inlet/reactor yielded charge states ranging from 7 to 4 + , maximizing in abundance at the [M + 6HI6+ ion. After reaction with D E A at a flow rate of approximately 3 X g/s, only the 4 + and 5 + charge states were observed in our apparatus ( m / z limit 2000), with the 4 + ion about 5 times as abundant as the 5 + ions. Reduction of the 3 cysteine-cysteine disulfide bonds yielded an ESI mass spectrum with charge states rang-
+
ing from 8 + to 4 + before D E A reaction (maximum intensity at 6 + to 7 + ) and 6 + to 4 + after reaction (maximum intensity at 5 +). Here also, reaction products were examined under a variety of conditions, but the charge-state shift in the disulfide-reduced species never equalled or exceeded that of the non-reduced species. It would be very exciting and potentially useful if the differences in reaction rate between the disulfidereduced and non-reduced proteins reflected the retention of significant portions of the solution conformation. However, the observed reaction rate differences could simply reflect the comparison between a molecule which is fully extended vs a molecule which, while also significantly extended, is still restrained by disulfide bonds at certain locations. On the basis of these studies, we can only conclude that the proton-transfer reactivities of equally charged reduced and nonreduced protein ions differ. From the viewpoint of protein collision cross-sections, the greater reactivity observed for individual charge states in the non-
+ 100
0
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7)
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8 0
.d
u
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.d
m
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mlz Figure 4. ESI-MS of native bovine albumin (M,66 430) (a) without and (b) with diethylamine. Capillary outletlskimmer bias 350 V. Capillary temperature 175 "C.The sample was dissolved in 5% glacial acetic acid+water and sprayed with a methanol sheath solvent.
GAS PHASE ION/MOLECULE REACTIONS WITH DIETHYLAMINE
164
exert more and more of an effect with increasing charge. It is also possible that the higher order structure of the ions may be altered by the high concentrations of amine reactant or by thermal processes during transit through the heated capillary inletheactor. Finally, since it is known that solution conformation can dramatically affect the pK,'s of the individual amino acid constituents of proteins:' it is possible that ion/ molecule chemistry could also probe higher order structural effects arising from weak (e.g., hydrogen bonding) interactions. Additional ESI investigations exploring these issues are being pursued. CONCLUSION A capillary interface/reactor has been used to study ion/molecule proton-transfer reactions of multiply protonated proteins generated by ESI. The disulfidereduced protein ions studied here appear to be substantially less reactive to proton transfer than 'native' protein ions with the same number of charges. In the case of proton-transfer reactions, it is expected that the increased Coulombic forces arising from different comformations and charge locations (i.e., specific charge sites and their proximities) should increase the rate and exothermicity of proton transfer from multiply charged proteins. Further studies aimed at understanding the differences in reactivity and the structure and chemistry of multiply charged macro-ions in the gas phase are in progress.
Acknowledgements
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We acknowledge W. H. Robins and C. G. Edmonds for helpful discussions, and the US Department of Energy, Office of Health and Environmental Research, through internal Exploratory Research of the Molecular Science Research Center (Contract DE-AC06-76RLO 1830) for support of this research. Pacific Northwest Laboratory is operated by Battelle Memorial Institute.
2000
mlz Figures. ESI-MS of reduced bovine albumin (a) without and (b) with diethylamine. Capillary outlet/skimmer bias 350 V. Capillary temperature 175 "C. The sample was dissolved in 5% glacial acetic acid water and sprayed with a methanol sheath solvent.
+
reduced proteins might be surprising, since the more extended structures expected for the disulfide-reduced proteins should result in larger collision cross-sections. However, the proton-transfer reactions relevant here occur at rates far below their collision rate.= When the Coulombic forces arising from charge repulsion are also considered, it seems likely that a highly charged, nonreduced protein would be more reactive than an equally charged reduced protein, because the charge states would most likely be spaced more closely in the more constrained conf~rmation.~' In such instances, the increased Coulombic forces should increase the rate and exothermicity of proton transfer from multiply charged proteins. There are several other considerations worthy of further investigation. We have considered protein conformation as two cases-native and denatured (i.e., reduced), but our analysis has not incorporated the possibility that the gas-phase conformation may vary substantially with charge state as Coulombic forces
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Allied Topics, pp. 266-267, Nashville, TN, ASMS, East Lansing (1991). 27. B. E. Winger, K. J. Light and R. D. Smith, J. A m . SOC.Mass Spectrom. (submitted). 28. R. R. Ogorzalek Loo, H. R. Udseth and R. D. Smith (in preparation). 29. S. K. Chowdhury, V. Katta and B. T. Chait, Rapid Commun. Mass Spectrom. 4, 81 (1990). 30. J. A. Loo, H. R. Udseth and R. D. Smith, Rapid Commun. Mass Spectrom. 2, 207 (1988). 31. C. J. Barinaga, C. G. Edmonds, H. R. Udseth and R. D. Smith, Rapid Commun. Mass Spectrom. 3, 16 (1989). 32. R. D. Smith, C. J. Barinaga and H. R. Udseth, Anal. Chem. 60, 1948 (1988). 33. A. L. Rockwood, M. Busman, H. R. Udseth and R. D. Smith, Rapid Commun. Mass Spectrom. 5 , 582 (1991). 34. M. Busman, A. L. Rockwood and R. D. Smith J. Phys. Chem. (in press). 35. W. W. Cleland, Biochemistry 3, 480 (1964). 36. C. C. McDonald, W. D. Phillips and J. D. Glickson, J . A m . Chem. SOC.93, 235 (1971). 37. J. Caslavska, P. Gebauer and W. Thormann, J. Chromatogr. 585, 145 (1991). 38. A. L. Rockwood, M. Busman and R. D. Smith, Int. J . Mass Spectrorn. Ion Processes (in press). 39. E. D. Anderson, W. J. Becktel, F. W. Dahlquist, Biochemistry 29, 2403 (1990).
