Anal. Chem. 2003, 75, 1599-1603
Plasma Electron Capture Dissociation for the Characterization of Large Proteins by Top Down Mass Spectrometry Siu Kwan Sze, Ying Ge, HanBin Oh, and Fred W. McLafferty*
Department of Chemistry and Chemical Biology Cornell University, Ithaca, New York 14853-1301
For structural characterization of multiply charged protein molecular cations by mass spectrometry (MS),1 conventional methods such as collisionally activated dissociation (CAD)2 or infrared multiphoton dissociation (IRMPD)3 that increase the ion
internal energy produce relatively similar MS spectra, cleaving the weakest interresidue -CO-NH- bonds to produce b, y fragment ions. For electrospray ionization (ESI) with Fourier transform (FT) MS, the new electron capture dissociation (ECD)4-7 is far less selective because of its far higher energy deposition (∼6 eV) and nonergodic time scale (n electrons than to another such ion. Thus, sufficient thermalization (a net velocity difference close to zero) should allow efficient electron capture. The Figure 2 cleavage map of carbonic anhydrase shows 237 different a•, b, c, y, and z• product ions; their total abundance represents 87% of the abundance decrease of the molecular ions. Normal ECD requires minimal electron kinetic energy; raising the electron energy by 1 eV reduced the ion capture cross section to ∼0.1% of its value.4 However, under these plasma ECD conditions, the electron kinetic energy from 150 (183 in Figure 2) cleavages at 8 V. One of the first experimental observations in this study was that a pulse gas cell pressure of ∼10-7 Torr (gauge reading) gives substantially better ECD spectra than does that of the ∼10-6 Torr generally used for normal ESI spectra to achieve translational ion cooling, although the 10-7 Torr conditions give satisfactory cooling, allowing ∼70% of the ions to pass through the cell untrapped. At lower pressure, the accelerating ion will have traveled farther into the cell before its first collision and will thus have gained more energy in addition to that required to overcome the front trap potential. Plasma ECD also enhances denaturation by minimizing the time for refolding.12 In the example above at low collision energies (5 V front trap), 116 cleavages resulted from electrons introduced during the first 0.67 s of ion introduction, but only 25 cleavages resulted from electrons introduced during 1.33-2.0 s, a time found sufficient for extensive refolding in smaller proteins.8 Minimizing Secondary Dissociation of Large Products Ions. However, under nonplasma AI ECD conditions, such higher collision energies did not produce spectra with an increased number of cleavages.7 To investigate this difference, collisionally activating the carbonic anhydrase ions under the same conditions as the 183 cleavage map (+8 V front trap, Figure 2), but instead adding the electrons just after the ion introduction, and in excess (0.3 µA of 90% of the molecular ions. The abundance of small terminal product ions has been greatly increased at the expense of the larger fragment ions, consistent with multiple secondary electron captures at denatured sites. These sites are also starting to refold;12 delaying eintroduction until 10 s after ion introduction gave the Figure 3B map of 105 cleavages from dissociation of 70% of molecular ions. Although the energetic (g8 eV) molecular ion collisions have also produced extensively denatured fragment ions, the plasma conditions that yield 83% molecular ion dissociation (Figure 2) with 87% efficiency have somehow produced far less fragment ion dissociation, especially that of the more highly charged fragment ions. As a possible explanation, consider the average electron behavior (Figure 1); these will travel in along the magnetic z-axis, accelerated by the +9 V on the back trap electrode. On entering the pressurized cell, they will be slowed by collisions and by the potential drop, so that most will not reach the grounded center of the cell. With the emitter on, electrons will be collisionally thermalized and accumulate in the cell, at least originally in the half where they enter. When the incoming electron beam is shut Analytical Chemistry, Vol. 75, No. 7, April 1, 2003
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Table 1. Charge-State Distribution of Molecular Ion Abundances before and after ECD nonplasma
Figure 3. Nonplasma ECD cleavage map under the conditions of Figure 2, except ion introduction was followed by 0.3-µA electron introduction (A) immediately and (B) with 10-s delay.
off, the thermalized electrons will tend to exit the cell along the z-axis toward the +9-V back electrode, with few toward the +8-V front electrode. Then the molecular ions, also collimated along the magnetic z-axis, are allowed to enter through the +8-V front trap electrode. In their acceleration toward the grounded cell center, the ions will undergo denaturing collisions, with few electrons present and these traveling in the opposite direction. However, between the cell center and the +9-V back electrode, the ions will be retarded in the area where the electrons reverse directions and are reaccelerated (Figure 1), making probable their juxtaposition with near-zero difference in velocity, as required for efficient e- capture. This will form product ions with nearly the same velocity and directionality of the precursor ions, but their continuing retardation will produce an increasing velocity differential versus the accelerating electrons; this should lower the probability of e- capture by the product ions. Those remaining will reverse direction before reaching the +9-V back electrode, traveling against the outbound electrons until the ions pass the cell center where they will only be exposed to few electrons of unfavorable capture velocities exiting toward the +8-V front trap electrode. Thus, the dynamics of the ECD plasma could account for the far more favorable electron capture by the incoming molecular ions than by their product ions. An additional effect appears to cause secondary dissociations of large product ions to be less favored, versus smaller products, than in conventional AI ECD (Figure 3A). Reflecting in part the 1602 Analytical Chemistry, Vol. 75, No. 7, April 1, 2003
plasma
charge
before
after
before
after
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
12 15 20 35 50 77 99 74 101 128 135 169 193 253 217 156 153
10 12 16 19 21 17 24 17 23 29 31 26 21 12 10 15 12
126 151 114 149 280 473 189 414 264 318 159 84 96 79 22 24 19
33 42 52 52 51 47 34 33 36 37 27 20 18 9 6 5 5
expected square dependence of charge state on e- capture cross section,4 ECD of carbonic anhydrase under nonplasma conditions reduces the 19+ to 26+ molecular ion abundance by 64% and that of the 28+ to 35+ ions by 89% (Table 1). However, the plasma conditions give 82% and 84% reductions, respectively. Apparently the nonequilibrium plasma conditions greatly reduce the high charge-state selectivity by making an excess of electrons available. To the extent that this is true for secondary e- capture of fragment ions, this would be a substantial advantage; for conventional AI ECD, a 15+ fragment ion representing half of carbonic anhydrase would have 25 times the cross section for e- capture versus that of a 3+ fragment ion of the terminal 10% of the protein, at least in qualitative agreement with the differences of Figures 2 and 3A. Although e- capture at the molecular ions of far higher charge state should also not be favored by the charge value squared, the plasma conditions that intermingle the electrons and ions still produce a high efficiency of molecular ion e- capture. Extent of Protein Characterization by Plasma ECD. Of the more than 100 ECD spectra measured here under plasma conditions, dozens exhibited >150 cleavages, substantially more than the highest 138 cleavages of our recent study.7 However, these many spectra gave cleavages at relatively similar backbone positions. The Figure 2 map with 183 cleavages used an +8-V front trap ion acceleration and 1-s ion introduction, while that with a 2-s introduction showed 156 cleavages. These two spectra combined showed 197 different cleavages, or 76% of the protein’s interresidue bonds. However, none of the other spectra gave as many as five new cleavages, although a total of 250 of the possible 258 were achieved in 25 nonplasma ECD spectra, measured under a wide variation of conditions.7 The unusual volume of data can also be valuable for top down protein characterization. The 183 cleavages in the Figure 2 spectrum of carbonic anhydrase actually represent 237 different a•, b, c, y, and z• fragment ions based on the mass values assigned to 512 isotopic clusters by THRASH;15 obviously many fragment ion m values are measured (m/z) in more than one charge state (z). However, 45 of the 512 mass values were 1 Da lower (peaks with asterisks, Figure 2) than those predicted by the sequence information in the NCBI protein database.17 Further, all 45 mass
Figure 5. Fragmentation map of β-casein from several plasma ECD spectra. Figure 4. Plasma ECD cleavage map of ubiquitin, +7-V front trap electrode.
values represent fragments from cleavages between residue 10 (Asp) and residue 31 (Asn). The molecular mass of aspartic acid is 1 Da heavier than asparagine, so that plasma ECD has provided highly convincing data that, indeed, the correct assignments are Asn-10 and Asp-31 for bovine carbonic anhydrase. The database also has these assignments for human and sheep carbonic anhydrase.17 Our earlier description of the top down approach to protein characterization used bovine carbonic anhydrase as a model protein but did not detect this error by MS/MS using CAD and IRMPD.9 Comparative proteolysis studies did find the -1-Da difference in some peptide masses; embarrassingly, this was attributed to deamidation during proteolysis. Plasma ECD of Small Proteins. Conventional ECD of ubiquitin (8.6 kDa) using separate trapping of the electrons and ions gave peaks representing cleavages of 67 of its 75 interresidue bonds as the most in one spectrum.4c Plasma ECD of the same protein with +8 V on the front trap cleaved 70 of its 75 interresidue bonds, while a +7 front trap resulted in 73 cleavages (Figure 4). Repeating the experiment, but instead adding the electrons and ions together, gave 72 cleavages including the two missing in Figure 4; prior denaturation of the protein ions is a far less serious problem for a 8.6-kDa versus a 29-kDa protein. Previously several ECD and CAD spectra were required to sequence ubiquitin de novo,18 but with plasma ECD this can be achieved with two spectra. Characterizing Posttranslational Modifications. Energetic ion dissociation methods such as CAD and IRMPD often break off amino acid modifications from the protein in competition with its backbone cleavage.19 The latter is heavily favored in ECD,5a which for phosphorylation was illustrated by the 24-kDa bovine (17) http://www.ncbi.nlm.nih.gov/. (18) Horn, D. M.; Zubarev, R. A.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10313-10317. (19) Carr, S. A.; Huddleston, M. J.; Annan, R. S. Anal. Biochem. 1996, 239, 180192. (20) Shi, S. D.-H.; Hemling, M. E.; Carr, S. A.; Horn, D. M.; Lindh, I.; McLafferty, F. W. Anal. Chem. 2001, 73, 19-22.
casein20 that contains 26 possible sites (Ser, Thr, or Tyr). In the original study with conventional AI-ECD, 87 of 208 backbone bonds were cleaved.20 Of the five actual phosphorylations, ECD pinpointed the site of one (Ser-15) and localized three more to be within five sites, and the last to within four sites. The plasma ECD fragment ion data (Figure 5) locate all five phosphorylation sites exactly from the total of 126/208 cleavages. Further, cleavages occur between all the remaining 21 Ser, Thr, or Tyr sites, so that any new phosphorylation should also be specified exactly in the corresponding ECD spectra. CONCLUSIONS Thermalizing the electrons and then the ions in a gas pulse intersperses the translationally cooled reactants to provide high ECD efficiency, while the original ECD technique only trapped the electrons and ions in separate adjacent groups. For larger proteins with extensive tertiary structure such as carbonic anhydrase, higher energy (e.g., g8 eV) collisions with the plasma gas provide products from the cleavage of as many as 183 of its 258 interresidue bonds in one spectrum and of 197 bonds in two spectra. The nonequilibrium plasma conditions substantially lower the probability for secondary dissociation of the large fragment ions. It may be possible to affect ECD with molecular ions that are trapped and mass selected by accelerating the ions within the cell by oscillating trapping voltages before the gas pulse and electron admission. With pulsed gas introduction, plasma ECD should be applicable to FTMS instrumentation without extra electron trapping electrodes, and possibly to ion trap and triple quadrupole instruments, greatly increasing the power and convenience of the top down MS/MS approach to biomolecular structural characterization. ACKNOWLEDGMENT We thank Kathrin Breuker, Barry Carpenter, Vlad Zabrouskov, and Huili Zhai for valuable discussions, and the National Institutes of Health (Grant GM16609) for generous funding. Received for review July 9, 2002. Accepted December 23, 2002. AC020446T
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