Anal. Chem. 1883, 65, 645-649
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Collisionally- Activated Dissociation of Multiply Charged 150-kDa Antibody Ions Rong Feng' and Yasuo Konishi National Research Council of Canada, Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada Electrospray (ES)1-4and matrix-assisted laser desorption6 ionization methods have greatly expanded the analytical applicability of mass spectrometry (MS)to large biomolecules. ES-MS,which offers high measurement precision6 and has an analytical mass range of -200 OOO Da for proteins? has been used to study protein conformational changes? enzyme reaction intermediates: inhibition mechanisms,1° covalentll and noncovalentl2 enzyme complexes, proenzyme maturation mechanism and kinetica,13 and the number of disulfidebonds14 in proteins. However, electrospray ionization itself provides little information about the structural connectivity of a
molecule, as this gentle ionization technique does not cause molecular fragmentation. Thus, collisionally-activated dissociation (CAD115becomes necessary for MS/MS16 structural analysis of electrospray-produced ions. The multicharging feature of protein ions has opened an exciting new channel for the CAD study of large biomolecules.17 Partial sequence information has been obtained from multiply charged ions of proteins as large as 66 demonstrating the feasibility of direct MSIMS sequencing for large proteins. This is in contrast to CAD of singly charged ions, where the fragmentation efficiency drops drastically with increasing ion mass, confiiing typical MS/MS analysis to the mass range of -3000 Da.18 Therefore, CAD of multiply charged ions hae the (1)Dole, M.; Mack, L. L.; Hines, R. L.; Mobley, R. C.; Ferguson, L. D.; Alice, M. B. J. Chem. Phys. 1968,49,2240-2249. potential to mature as a technique for direct structural analysis (2)(a) Wong, S.F.; Meng, C. K.; Fenn, J. B. J.Phys. Chem. 1988,92, of large biomolecules. In keeping with the ever increasing 546-550. (b)Fenn,J.B.;Mann,M.;Meng,C.K.;Wong,S.F.;Whitehouse, sizes of protein ions produced by the electrospray method, C. M. Science 1989,246,64-71. (c) Fenn, J. B.; Mann, M.; Meng, C. K.; probingthe feasibility of gas-phasefragmentation of multiply Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990,9,37-70.(d) Mann, M. Org. Mass Spectrom. 1990,25,575-587. charged ions of higher masses becomes necessary. We report (3)(a) Loo, J. A.; Udseth, H. R.; Smith, R. D. Anal. Biochem. 1989, here the CAD study of multiply charged 150-kDa antibody19 179,404-412. (b) Smith, R.D.; Loo, J. A,; Edmonds, C. G.; Barinaga, C. ions using a triple-quadrupole20 mass spectrometer. J.; Udseth, H. R. Anal. Chem. 1990,62,882-899. (c) Smith, R.D.; Loo,
J. A.; Ogorzalek Loo, R. R.; Busman, M.; Udseth, H. R. Mass Spectrom. Rev. 1991,10,359-452. (4)(a) Bruins, A. P.; Covey; T. R.; Henion, J. D. Anal. Chem. 1987,59, 2642-2646. (b) Covey, T. R.; Bonner, R. F.; Shushan, B. I.; Henion, J. Rapid Commun. Mass Spectrom. 1988, 2, 249-256. (c) Bruins, A. P. Mass Spectrom. Rev. 1991,10,53-77. (5)(a)Karas,M.;Bahr,U.;Ingendoh,A.;Hillenkamp,F.Angew. Chem., Int. Ed. Engl. 1989,28,760-761. (b) Karas, M.; Bahr, U.; Giessmann, U. Mass Spectrom. Rev. 1991,10,335-357. (c) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991,63,1193A-1203A. (6)(a) Henry, K.D.; Quinn, J. P.; McLafferty, F. W. J.Am. Chem. SOC. 1991,113,5447-5449. (b) Feng, R.; Konishi, Y.; Bell, A. W. J. Am. SOC. Mass Spectrom. 1991,2,387-401. (7) Feng, R.; Konishi, Y. Anal. Chem. 1992,64,2090-2095. (8) (a) Chowdhury, S. K.; Katta, V.; Chait, B. T. J. Am. Chem. SOC. 1990,112,9012-9013. (b) Katta, V.; Chait, B. T. J.Am. Chem. SOC. 1991, 113,8634-8535. (c) Loo, J. A.; Ogorzalek Loo, R. R.; Udseth, H. R.; Edmonds, C. G.; Smith, R. D. Rapid Commun. Mass Spectrom. 1991,5, 101-105. (d)LeBlanc,J.C.Y.;Beuchemin,D.;Siu,K.W.M.;Guevremont, R.; Berman, S. S. Org. Mass Spectrom. 1991,26,831-839.(e) Feng, R.; Konishi, Y. Proceedings of the 39th ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, May 19-24, 1991; American Society for Mass Spectrometry: East Lansing, MI, 1991;pp 1432-1433. (9)(a) Stevenson, D. E.; Feng, R.; Storer, A. C. FEBS Lett. 1990,277, 112-114. (b) Stevenson, D. E.; Feng, R.; Dumas, F.; Groleau, D.; Mihoc, A.; Storer, A. C.Biotechnol. Appl. Biochem. 1992,15,283-302.(c) Aplin, R.T.;Baldwin, J. E.; Schofield, C. J.; Waley, S. G.FEBSLett. 1990,277, 212-214. (d) Shneier, A.; Kleanthous, C.; Deka, R.; Coggins, J. R.; Abell, C. J.Am. Chem. SOC. 1991,113,9416-9418. (IO)MBnard, R.; Feng, R.; Storer, A. C.; Robinson, V. J.;Smith, R. A.; Krantz, A. FEBS Lett. 1991,295,27-30. (11)Feng, R.; Yuan, 2.Proceedings of the 38th ASMS Conference on Mass Spectrometry and Allied Topics, Tucson, AZ, June 3-8, 1990; American Society for Mase Spectrometry: East Lansing, MI, 1990; pp 998-999. (12)(a) Ganem, B.;Li, Y.-T.; Henion, J. D. J.Am. Chem. SOC. 1991, 113,6294-6296. (b) Ganem, B.; Li, Y.-T.; Henion, J. D. J. Am. Chem. Soc. 1991,113,7818-7819.(c) Baca, M.; Kent, S.B. H. J.Am. Chem. SOC. 1992,114,3992-3993. (13)Rowan, A. D.; Feng, R.; Konishi, Y.; Mort, J. S. Biochem. J., submitted. 0003-2700/93/0365-0645$04.00/0
EXPERIMENTAL SECTION Murine anti-(human q-acid glycoprotein) monoclonal antibody, an immunoglobulin of GI subclass (IgGl), was obtained from Calbiochem Corp. (SanDiego, CA). The stabilizing buffer and inpurities in the originalsample were removed by centrifugal ultrafiltration through a YM-membrane (Amicon,Beverly, MA) having a molecular weight (MW)cutoff of 100 OOO. The filtration procedure was repeated three times to ensure a high sample purity (14)Feng, R.; Bell, A.; Dumas, F.; Konishi, Y. Proceedings of the 38th ASMS Conference on Mass Spectrometry and Allied Topics, Tucson, AZ, June 3-8, 1990; American Society for Mass Spectrometry: East Lansing, MI, 1990;pp 273-274. (15)(a) Haddon, W. F.; McLafferty, F. W. J.Am. Chem. SOC. 1968, 90, 4745-4746. (b) Jennings, K. R. Znt. J. Mass Spectrom. Zon Phys. 1968,1,227-235. (c) McLafferty, F. W.; Bente, P. F., III; Kornfeld, R.; Tsai, S.-C.; Howe, I. J. Am. Chem. SOC. 1973,95,2120-2129. (16)(a) McLafferty, F. W. Science 1981,214,280-287. (b) Tandem Mass Spectrometry; McLafferty, F. W., Ed.; Wiley: New York, 1983.(c) Busch, K. L.; Glish, G. L.; McLuckey, S. A. Mass SpectrometrylMass Spectrometry: Techniques and Applications of Tandem Mass Spectrometry; VCH New York, 1988. (17)(a) Loo, J. A.; Quinn, J. P.; Ryu, S.I.; Henry, K. D.; Senko, M. W.; McLafferty, F. W. h o c . Natl. Acad. Sci. U.S.A. 1992,89,286-289. (b) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Anal. Chem. 1991,63,24882499. (c) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Science 1990,248, 201-204. (d) Loo,J. A.; Edmonds, C. G.; Udseth, H. R.; Smith, R. D. Anal.Chim.Acta 1990,241,167-173.(e)Smith,R.D.;Loo, J.A.;Barinaga, C.J.; Edmonds, C. G.; Udseth, H. R. J. Am. SOC. Mass Spectrom. 1990, 1,5345.(0Smith, R. D.;Barinaga, C. J. Rapid Commun.Mass Spectrom. 1990,4,54-57. (18)(a) Biemann, K. In Biological Mass Spectrometry; Burliigame, A. L., McCloskey, J. A., Eds.; Elsevier: Amsterdam, 1990;p 182. (b) Hayes, R. N.; Gross, M. L. In Methods in Enzymology, Vol. 193,Mass Spectrometry; McCloskey,J. A., Ed.; Academic Press: San Diego, 1990; pp 237-263. (c) Alexander, A. J.;Boyd, R. K. Int. J.Mass Spectrom. Ion Processes 1989,90, 211-240. (d) McLuckey, S. A. J. Am. SOC. Mass Spectrom. 1992,3, 599-614.
Published 1993 by the American Chemlcal Soclety
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for the MS study. Horse heart cytochrome c was obtained from Sigma Chemical Co. (St. Louis, MO) and used without further purification. Both the IgG antibody and cytochrome c were dissolved in pure water with 10%acetic acid (v/v, pH 2.2), at the and 8 X 104 M, respectively. concentrations of 3 X A triple-quadrupole mass spectrometer (the API I11 MS/MS system, Sciex, Thornhill, Ontario, Canada) was used for the ES and MS/MS studies. The construction of the instrument is essentially the same as the one depicted in ref 20c, except that the original postorifice lens system has been replaced with a quadrupole ion guidance lens. The mass-to-charge (mlz)range of the instrument has been expanded to 2400 and the ionization chamber fitted with a pneumatically-assisted electrospray (also referred to as ionspray)4device. Multiply charged protein ions were generated by spraying the aqueous sample solution into the ambient air through a stainless steel capillary held at +5.2 to +5.5 kV above the instrument ground potential. A coaxial air flow (0.6L/min) along the capillary sprayer was provided to assist the liquid nebulization; the nebulizer pressure was set to 20 psi. The sample solution was delivered, at a flow rate of 0.5 gL/min for the antibody and 1.0 gL/min for cytochromec, to the sprayer by a syringeinfusionpump (Model22, Harvard Apparatus, South Natick, MA) through a fused silica capillary of 100-gumi.d.; the low flow rates were chosen to reduce the sample consumptions. The ion entrance orifice (- 100-pmdiameter) at the atmosphere/ vacuum interface of the instrument was +140 V biased against in front of the first quadrupole the quadrupole guidancelens (QO) A gas curtain formed by a continuous flow mass analyzer (Q1). (1.2 L/min) of nitrogen gas around the conical orifice served to prevent the moisture from entering the orifice and to cause multiple desolvationcollisionsin the postorifice free-jetexpansion region and in the QOquadrupole guidance lens. Unless otherwisestated, in the antibody MS/MS experiments all ions of various charge states, exiting from the Q1mass analyzer (designated as MS-1) without m/zseparation, were subjected to collisions at various acceleration voltages with argon collision with gas in the 15-cm-long quadrupole collision chamber (Qz, only radio-frequency potentials20), and the product ions were then m/z analyzed by the second quadrupole mass analyzer (Q3, designated as MS-2). A retarding potential, biased against the QOguidance lens by +30 V, was applied to the Q1mass analyzer to slow down the incoming antibody ions, which resulted in an intensity reduction of the precursor ions by -20%. The Q2 collision chamber was negatively biased against both Q1 and Q3 mass analyzers to first accelerate the precursor ions for collisions and then decelerate the product ions for m/z analysis in Q3.The ion acceleration voltage, i.e., the bias potential between Q1and Q2,was varied from 80 to 160 V for various CAD experiments, unless otherwise stated. An argon collision gas thicknesslMof 4.5 X 1014atoms/cm2 along the Q2axis, which was derived by integrating the gas density distribution from the free-jet expansion of the collision gas perpendicular to the ion path,z0"was employed in the antibody CAD experiments. The collision scatteringldissociation losses, and the poorer transmission efficiencyof the Q2collisionchamber at the increased bias voltages, reduced the precursor ion intensity by 80 % .
-
RESULTS AND DISCUSSION Figure l a is the electroapray maas spectrum of murine anti(humanal-acid glycoprotein)*l monoclonal antibody (average MW 149 600)7in the absence of dissociative collisions. Only the partialspectrum (m/z1200-2400) containiigthe antibody (19)(a) KBhller, G. Science 1986, 233, 1281-1286. (b) Milstein, C. Science 1986,231,1261-1268.(c) Amzel, L.M.; Poljak, R. J. Annu. Rev. Biochem. 1979,48,961-997.(d) Lehninger, A. L. Biochemistry, 2nd ed.; Worth New York, 1975; p 1003. (e) Putnam, F. W. In The Plasma Proteins, Vol. 5 , Structure, Function, and Genetic Control, 2nd ed.; Putnam, F. W., Ed.; Academic Press: Orlando, FL, 1987;pp 49-140. (20)(a)QuadrupoleMassSpectrometryandltsApplications; D a m n , P. H., Ed.; Elsevier: Amsterdam, 1976. (b) Yost, R.A.; Boyd, R. K. In Methods in Enzymology, Vol. 193,Mass Spectrometry; McCloskey, J. A., Ed.; Academic Press: San Diego, 1990,pp 154-200. (c) Dawson, P. H.: French. J. B.; Bucklev, J. A.;. Douglas, D. J.; Simmons, D. Org. Mass Spectrom. .1982,17, 205-211. (21)Fraevman, N. H.; De Smet, F. H.: Van De Velde, E. J. Hybridoma 1987,6,565I-574.
Rei. Int. (YO)
63'
I
1
loo
100
1
100 1
"
I
1500
2000
m/Z
Flgure 1. The electrospray mass spectrum (a) and the CAD spectra (b-d) of murlne anwhuman al-add gtycoproteln)monoclonalantibody (average MW 149 600) obtained on a trlple-quadrupole mass spectrometer. Lower MW mtamlnants, which mlght Interfere with the fragment ion signals, were not registered In the mlz range scanned (mlz100-2400 scanned, only mlz 1200-2400 shown). To recordthe total charge-state CAD spectra shown In panels b-d, all antibody ions of various charge states (63+ to 74+), wlthout mlz separation by MS1, were Injected Into the quadrupole collision chamber to coUlde with argon at a collision gas thldtness of 4.5 X loi4 atomslcm*, and the acceleratlon voltage was varied from 80 to 160 V as Indlcated. MS2 detected no appreciable signals of fragment ions bebw mlz 1200. I n panels c and d the llght-chaln fragmnt (23 720 i 30 Da) Ions are marked by their charge states (114- to 14+). The number of scans and the sample consumptbns In the spectra shown were (a) 30 scans and 22 pmol, (b) 25 scans and 18 pmol, (c) 35 scans and 25 pmol, and (d) 45 scans and 32 pmol.
signalsis shown; the full spectrum, scanned down to mlz 100, registered no appreciable signals of contaminating impuritiea. The antibody charge distribution profile waa truncated because the quadrupole maas spectrometer, having an m/z range of 2400, could record only the high charge-state ions (63+ to 74+). T o obtain the CAD spectra shown in Figure lb-d, MS-1 waa run under the total-ion mode, i.e, with only radiofrequency potentials applied on &I, to simultaneously transport all antibody ions of various charge statesto the QZcollision chamber. One of the anticipated advantages of this "multichannel" approach waa the senaitivity gain for the fragment ions, aa all precursors contribute simultaneously to the CAD spectrum. This expectation waa borne out by the fact that only tens of picomoles (10-12 mol) of samples were consumed to obtain the CAD spectra shown in Figure lb-d, which were
ANALYTICAL CHEMISTRY, VOL. 65, NO. 5, MARCH 1, 1903 24 kDa
51 kDa
51 kDa
24 kDa
647
Rel. Int. (%)
25m0i
825 617 649 1
Flgure 2. Simplified schematlc structure of murine IgQl-subclass
antlbody (150 kDa) showing only the main polypeptide chains (thick Ilnes) and the Interchain dlsuHide brldges (thln lines). The two 24-kDa llght (L) and the two 51-kDa heavy (H) chains are llnked together In a Y-&ape by two Interl-Kchaln and three Inter-H-Kchaln dlsulflde brldges made of (C)CH2SSCH2(C)type of bond structure.
in the same sample consumption range for the normal electroepray spectrum shown in Figure la. Furthermore, it was hoped that the antibody fragment ions from this multichannel CAD would form a secondary charge-state distribution to help the interpretation of the CAD results, as the charge states of the precursors themselves were in a distribution to start with. This turned out also to be true, as shownby the fragment ion seriesin the CAD spectra (Figure lc,d). Since there were no interfering low MW contaminant ions in the electrospray mass spectrum (Figure la), the m/z selection of the antibody precursor ions prior to CAD was not a critical factor for the purpose of studying the fragmentation feasibility, as they differed only in their number of charges. To evaluate the effect of kinetic energy on ion fragmentation, the acceleration voltage was varied from 80 to 160 V in the CAD experiment. As seen in Figure lb-d, the CAD spectra showed a strong dependence of the fragment ion abundance on the collision energy. At 80-V accelerationmost of the antibody ions survived the collisions (Figure lb), although the ions had 5-6 keV of kinetic energy. However, raising the acceleration voltage to 130 V (ion kinetic energy 8-10 keV) increased substantially the fragment ion intensities (Figure IC).At 160-V acceleration (ion kinetic energy 10-12 keV), a large fraction of the antibody ions decomposed, as evidence by the intense fragment ion signals (Figure Id). Further increasing the acceleration voltage to 210 V (ion kinetic energy 13-16 keV) did not change the overall spectral pattern but reduced the signal-to-noise ratio of the fragment ions to only -3:1, due to the greatly decreased collection efficiency of the triple-quadrupole system at high bias voltage. The observed strong energy dependence between 80- and 160-V accelerationindicates that the antibody fragmentation took place near an energetic threshold. The secondary charge-state distribution in the antibody CAD spectra yielded3~4ba fragment mass of 23 720 f 30 Da and ion charge states of 11+to 14+. (The background at the high m/z end of the CAD spectra shown in Figure lb-d appears to be much higher than the one in the normal electrospray spectrum shown in Figure la, due to the reduction of the precursor signals by -80% in the collision chamber and the subsequent spectral intensity normalization.) The fragment maas is consistent with that of the light (L) chain (-24 kDa) of the antibody, indicating that the single interchain disulfide bridgelgh that links the L-chain to the heavy (H) chain (-51 kDa) has been collisionally ruptured (see Figure 2 for a schematic structure of the murine IgG1-subclass antibody). Destruction of a disulfide bridge, i.e., a bond structure of (C)CH&SCH&) type cross-linking two cysteine amino acid residues, has been shownz2to be a preferred collisional dissociation pathway of disulfide-bridged dimeric peptides. (22) Bean, M. F.; C a r , S . A. Anal. Biochem. 1992, 201, 216-226.
0 500
600
700
m,z 800
900
1000
Flgure 3. The electrospray CAD spectrum of the horse heart cytochrome c (MW 12 358)Ions carrying 15 protons (mlz 625). The Ions colllded with argon In the Q2 collision chamber at a colllsion gas thickness of 6.4 X lo1' atoms/cm2 and a translational energy of 870 eV (centerof-mass collklonenergy of 2.8 eV). The two slngly-charged major fragments at mlz 617 and 649, which differed In mas8 by 32 Da, were due to the colllslonal release of the heme group that was C-S bondedto the polypepWe chain. No other hlghlntensltyfragments were detected outside the scan range shown. The spectrum was the sum of 1000 scans and consumed 360 pmol of sample. Cytochrome c Ions of other charge states, run under the charge-selected CAD mode, producedthe heme fragments of slmkr pattern. Run under the total chargsstate CAD mode, with all ions from 6-t to 20+ contributing to the spectrum, an lntenslty Increase of -6O-fdd was reglstered for the two maJor heme fragments.
Similarly, as shown in Figure 3, the collisional release of the heme group that is C-S bonded to the polypeptide chain of horse heart cytochrome c (MW 12 358) has also been found to be the predominant fragmentation pathway at low collision energy (at high collision energy the protein dissociates mostly via other backbone cleavage^'^^). These CAD results can be rationalized qualitatively on the basis of known neutral bond strengths in a protein. (However, the large number of charges, believed to be located mostly on the side-chainamino groups of basic amino acid residues such as arginine, lysine, and histidine, may cause Coulombicstress and weaken the covalent bonds in multiply charged ionsz3). A disulfide bridge constitutes the weakest linkage across a polypeptide chain, as the dissociation energies248 of C-S (57 kcal/mol, or 2.47 eV) and S-S (72 kcal/mol, 3.12 eV) bonds are substantially lower than those of others (e.g., C-C bond, 82 kcal/mol, 3.56 eV;24aC(=O)-C, 81 kcal/mol, 3.51 eV;24b C-N, 84 kcal/mol, 3.64 and C(=O)-N, 101 kcal/mol, 4.38 eV24b). Therefore, the rate of disulfide splitting should be higher than those of other bond cleavages near the dissociation threshold. The peak widths of the L-chain fragment are broader than those of the antibody precursor, consistent with the high-resolution observationz2that triplet fragments, with a sequential mass increment of 32 Da, are produced in breaking a disulfide bridge which has three neighboring weak points at C-S, S-S, and S-C bonds. Breaking the inter-L-H-chain disulfidebridge would release two major fragments, a 24-kDa L-chain and its 125-kDa complementary fragment made of one L- and two H-chains. However, the 126-kDa fragment was not detected, neither was the 102-kDa fragment that would come from losing two L-chains. The reasons for the discrimination against the higher mass fragments are still not clear. Insufficientnumber of charges on these larger fragments may have prevented them from being detected. For example, if the departing (23) (a) Rockwood, A. L.; Busman, M.; Smith, R. D. Znt. J. Mass Spectrom. ZonProcesses 1991,111,103-129. (b)Busman,M.;Rockwood, A. L; Smith, R. D. J. Phys. Chem. 1992,96,2397-2400. (24) (a) McMillen, D. F.; Golden, D. M. Annu. Reu. Phys. Chem. 1982, 33, 493-532. (b) Benson, S. W. Thermochemical K i n e t i c s ; Wiley-Interscience: New York, 1976; p 309.
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24-kDa L-chain takes away 11charges from the 150-kDa IgG ion of 63+, ita 126-kDa complementaryfragmentof 52+ would have an m/z value of 2421 (= (149 600 - 23 720)/(63 - l l ) ) , which is beyond the 2400 m/z range of the instrument. Collisionaldeprotonation would also exacerbate the problem. Therefore, an instrument having an m/z range higher than 2400 is highly desirable for more thorough CAD studies on macrosized biomolecules. The 75-kDa fragment, which consists of an L-chain and an H-chain and would come from splitting the Y-shaped 150-kDa IgG molecule in the middle, was also not observed. This appears to be in part due to the fact that the inter-H-H-chain linkage in a murine IgG1subclass antibody consists of three disulfide bridges13e (see Figure 2) whose destruction would demand a much higher energy input. Producing the 51-kDa H-chain fragment is even less probable, as it requires not only cleaving the three inter-H-H-chain disulfide bridges, but also one additional inter-L-H-chain disulfide bridge. At 160-V acceleration, the kinetic energies of these antibody ions (63+ to 74+) ranged from 10 to 12 keV. However, due to the large mass difference between the antibody and argon (40 Da), each impact had only up to 2.7-3.2 eV (average 3.0 eV, or 69 kcal/mol) center-of-mass collision energies1sH available to excite these multi-keV ions. In addition, the antibody had -66 000 vibrational degrees of freedom (based on 1320 amino acid residues19d and on average 16 atoms/ residue, and -33 carbohydrate units and on average -26 atoms/unit per IgG molecule) to dissipate the excitation energy. Apparently, to have an appreciable amount of ion fragmentation, many collisional encounters with the argon gas were needed to accumulate enough internal energy. By loosely applying the quasi-equilibrium theoryz5 (BET), one can estimate (assuming a frequency factor of 1013 s-l) that the internal energy needed to have observable dissociations for the weakest C-S bond (during the 10-5 s ion residence time in the collision chamber) is -9000 eV. Assuming at 160-V acceleration the antibody ions on average acquired 3.0-eV internal energy per collision, -3000 collisions would be needed to accumulate enough internal energy. Since the amount of internal energy deposited per impact is always less than the center-of-masscollision energy, the actual number of collisions required could be substantially higher than 3000. However, acquiring9000-eV internal energy would mean that 75-90% of the ion translational energy has been lost and the ions have been slowed down considerably. Furthermore, a calculation based on the upper-limit physical cross-section of -7000 A2 for the IgG antibody (estimated from the X-ray crystallographic data29 and the argon collision gas thicknesslM value of 4.5 X 1014atoms/cmzshows that an IgG antibody ion would encounter only -300 collisions. On the other hand, ions passing through the orifice region can be "heated up" substantially by a very large number of desolvation collisions with the nitrogen curtain gas, if the orifice voltage is raised sufficiently high to energize these ~ollisions.1~b-f~Z0~ In fact, when the orifice voltage was raised to the maximum available value, the antibody ions started to fall apart in the interface region (see Figure 4). In conducting the Q2 CAD experiments shown in Figure 1, a normal operating orifice voltage was applied. The extent of "preheating" in these antibody ions before entering the QZ collision chamber could not be exactly determined using the present experimental setup, as it was uncertain how many
-
-
-
(25) Rosenstock, H. M.; Wallenstein, M. B.; Wahrhaftig,A. L.; Eyring, R. h o c . Natl. Acad. Sci. U.S.A. 1962, 38,667-678. (26) (a) Sarma, V. R.; Silverton, E. W.; Davies, D. R.; Terry, W. D. J. Biol. Chem. 1971, 246, 3753-3759. (b) Silverton, E. W.; Navia, M. A.; Davies, D. R. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 5140-5144. ( c ) Papalian, M.; Lafer, E.; Wong, R.; Stoller, B. D. J.Clin. Invest. 1980,65, 469-477.
Rel. Int. (%)
1o+
100 1
\I
M
04 1200
I
l6O0
m/z
2000
2400
Flgure 4. The electrospray CAD spectrum of
murine anWhuman alacid glycoprotein) monoclonal antibody obtained at the atmosphere/ vacuum interface of the mass spectrometer by raising the bias voltage on the sampling orifice to Its maximum value of 250 V. The high orifice vottage energized a very iarge number of coiilslons encountered by the antibody ions during the free-jet expansion with the nitrogen curtein gas and caused the ions to dissociate prior to the m/z analysis. The iight-chain fragment assigments are the same as the ones shown in Figure 1. The spectrum, which consumed 71 pmoi of sample for accumulating 100scans, was recorded by scanning MS1 of the triplequadrupole instrument. ion-molecule collisions were involved in the atmosphere/ vacuum interface region, and it was difficult to correlate the orifice voltage with the actual ion acceleration and therefore the internal energy deposition, under the near-thermal collision conditions. However, one could reasonably assume that under the orifice conditions employed, the collisional preexcitation was moderate, considering the fact that the noncovalently-bound myoglobin-heme complex could be observed& under similar conditions. Nevertheless, these moderately preheated antibody ions would require less additional excitation energy, and consequently fewer Collisions with argon, to break down. The Coulombic destabilization caused by the large number of charges on the antibody ions could also lower the dissociation barrier,23 further reducing the energyrequirement. Due to these uncertainties, the above estimates for the IgG antibody should be viewed only as the upper limits of the collision parameters. The collisional interaction between an argon atom and an IgG antibody ion appears to be localized during the initial impact, due to the large difference between their physical cross-sections (a cross-section of -7 A2 is estimated for an argon atom, using the radius of 1+ argon ion in crystal latticez7). In light of the proposed1sw28"impulse or spectator" collisional activation model (which hypothesizes that only the immediate impact portion of a projectile interacts with a target via internuclear momentum transfer),it is reasonable to assume that some local "hot spots" may exist on the antibody ion if a complete statistical redistribution of the collisional excitation energy has not been reached before the fragmentation starta to occur. Nonstatistical redistribution of excitation energy has been observed in the collisional fragmentation23aof polyatomic organic ions and in the unimolecular dissocation29bof ion-molecule complexes. Conceivably, a dynamicallylocalized excitation can still rupture a weak bond (such as a C-S bond) in the vicinity, although as a whole the energy input to the ion has not reached the threshold. If the impulsive collision-activation mechanism also operates in the multi-keV CAD of these antibody ions, the total internal energy required to break a bond can be substantially lower. In the 5-12-keVcollision energy range employed it appears that mainly the destruction of the antibody's weak interchain (27) CRC Handbook of Chemistry and Physics, 71st ed.; Lide, D. R., Ed.; CRC: Boston, 1990; p 12-1.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 5, MARCH 1, 1003
disulfide bridge has occurred. Apparently, for more fragmentation a higher energy input is necessary. The means for achieving it is currently under study. As shown by the antibody CAD experiment in the atmosphere/vacuum interface region (Figure 4), a simple and effective remedy may come from collisionally preheating"b-fp20e these ions with a very large number of collisionsduring the free-jet expansion. On the other hand, a magnetic-sector type of instrument,30 where the kinetic energy of a 70+ ion would be 700 keV at 10-kV acceleration, may also provide more collisional excitation energy. For a quadrupole type of instrument, the newly demonstrated acceleration-deceleration collisional technique31appears promising for booatingthe ion collision energy. (28)(a) Singh, S.;Harris, F. M.; Boyd, R. K.; Beynon, J. H. Znt. J. Mass Spectrom.IonProcesses 1985,66,131-149. (b)Uggerud,E.;Derrick, P.J. 2.Naturforsch. 1989, M a , 245-246. (c) Homing, S.R.; Vincenti, M.; Cooks, R. G. J. Am. Chem. SOC.1990,112,119-126. (d) Alexander, A. J.; Thibault, P.; Boyd, R. K. J.Am. Chem. SOC.1990,112,2484-2491. (e) Alexander, A. J.; Thibault, P.; Boyd, R. K.; Curtis, J. M.; Rinehart, K. L. Int. J. Mass Spectrom. Zon Processes 1990,98,107-134. (29)(a) Douglae, D. J. J. Phys. Chem. 1982,86,185-191. (b) Graul, S.T.; Bowers, M. T. J. Am. Chem. SOC.1991,113,9696-9697. (30)(a)Allen, M. H.; Lewis, I. A. S. Rapid Commun.Mass Spectrom. 1989,3,255-258.(b) Gallagher, R.T.; Chapman, J. R.; Mann, M. Rapid Commun.Mass Spectrom. 1990,4,369-372.(c) Larsen, B.S.; McEwen, C. N. J. Am. SOC.Mass Spectrom. 1991,2,205-211. (31)Turecek, F.;Gu, M.; Shaffer, S. A. J. Am. SOC.Mass Spectrom. 1992,3,493-501.
649
CONCLUSIONS The antibody CAD study demonstrates that multiply charged macrosized ions as large as 150kDa can be fragmented by collisions with argon gas. Although with the present experimental setup the structural information generated is still limited, it is nevertheless encouraging to future MS/MS studies on macrosized biomolecules. For multiply charged IgG antibody ions, the gas-phase fragmentation is readily observable, despite the low acceleration voltage (-200 V) available on the triple-quadrupole mass spectrometer and the huge 'heat-sinking" capability of the antibody's -66 OOO vibrational modes. The internal energy required for the observable fragmentation appears to come from the many collisions encountered by the antibody ions. On the other hand, localized collisional interactions may also play a role and reduce the total amount of internal energy required.
ACKNOWLEDGMENT We thank our colleagues S. Wu, S.Y. Yue, M.Cygler, F. Bouthillier, and B. F. Gibbs, and B. Thomson of Sciex Inc., for helpful discusions and the reviewersfor suggesting various manuscript modifications. This is NRCC publication no. 33709. RECEIVED for review August 12, 1992. Accepted December 3, 1992.
