Deuterium Scrambling during Quadrupole Time-of-Flight

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Anal. Chem. 2007, 79, 153-160

Hydrogen/Deuterium Scrambling during Quadrupole Time-of-Flight MS/MS Analysis of a Zinc-Binding Protein Domain Peter L. Ferguson,† Jingxi Pan,† Derek J. Wilson,†,‡ Brian Dempsey,‡ Gilles Lajoie,†,‡ Brian Shilton,‡ and Lars Konermann*,†,‡

Department of Chemistry, The University of Western Ontario, London, Ontario N6A 5B7, Canada, and Department of Biochemistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada

It remains an open question as to whether experiments involving collision-induced dissociation (CID) can provide a viable approach for monitoring spatially resolved deuteration levels in electrosprayed polypeptide ions. A number of laboratories reported the successful application of CID following solution-phase H/D exchange (HDX), whereas others found that H/D scrambling precluded sitespecific measurements. The aim of the current work is to help clarify the general feasibility of HDX-CID methods, using a 22-residue zinc-bound protein domain (Zn-ZBD) as model system. Metal binding in Zn-ZBD should confer structural rigidity, and the presence of several basic residues should sequester mobile charge carriers in the gas phase. Both of these factors were expected to suppress the extent of scrambling. HDX was carried out by employing rapid on-line mixing, thereby mimicking conditions typically encountered in kinetic pulse-labeling studies. Quadrupole time-of-flight MS/MS of pulse-labeled ZnZBD provides high sequence coverage. However, the measured fragment deuteration levels do not correlate with the known H-bonding pattern of Zn-ZBD, suggesting the occurrence of extensive scrambling. Instead of showing a uniform distribution, the fragment ions reveal a distinct nonrandom pattern of deuteration levels. In the absence of prior information, these data could erroneously be ascribed to the presence of protected sites. However, the observed patterns clearly originate from other factors; possibly they are caused by modulations of the amide CID efficiency by kinetic isotope effects. It is concluded that scrambling does not represent the only conceptual problem in HDX-CID studies and that control experiments on uniformly labeled samples are essential for ruling out interpretation artifacts. Electrospray ionization mass spectrometry (ESI-MS) is a powerful tool for exploring protein folding and dynamics. The information obtained by this technique is complementary to that acquired by other methods such as NMR spectroscopy or X-ray * To whom correspondence should be addressed. E-mail: [email protected]. http://publish.uwo.ca/∼konerman/. † Department of Chemistry. ‡ Department of Biochemistry. 10.1021/ac061261f CCC: $37.00 Published on Web 11/22/2006

© 2007 American Chemical Society

crystallography.1 Many approaches for studying proteins involve the use of hydrogen-deuterium exchange (HDX). H atoms in N-H, O-H, and S-H bonds can be replaced with deuterium from a D2O-containing solvent. Exchange is drastically slowed down at sites that are protected, either sterically or by H-bonding. Therefore, the HDX behavior of a protein reflects its overall structure and conformational flexibility.2 Isotope labeling in HDXMS is normally terminated by acid quenching and cooling of the reaction mixture. Peptide mapping followed by LC/ESI-MS allows HDX patterns to be determined by measuring the mass shifts of individual proteolytic fragments. The spatial resolution of this approach is on the order of a few residues. Labeling studies can also be carried out with NMR detection, which provides average deuteration levels for individual exchangeable sites. However, the protein concentrations required for NMR are at least 2 orders of magnitude higher than for MS. Moreover, NMR has a practical size limit of ∼30 kDa, whereas MS can routinely be used for the analysis of much larger proteins. As a result, the use of HDX-MS is rapidly gaining in popularity.3-7 A question that is currently receiving considerable attention is whether collision-induced dissociation (CID) in the gas phase can provide an alternative strategy for extracting site-specific HDX information.8,9 This type of ion activation is widely accessible and easily implemented on different types of mass analyzers. CIDHDX studies can be carried out in a “top-down” fashion that involves the fragmentation of whole proteins, thereby circumventing the use of solution-phase peptide mapping.10 CID has also been used in order to enhance the spatial resolution in classical (1) Kaltashov, I. A.; Eyles, S. J. Mass Spectrometry in Biophysics; John Wiley and Sons, Inc.: Hoboken, NJ, 2005. (2) Krishna, M. M. G.; Hoang, L.; Lin, Y.; Englander, S. W. Methods 2004, 34, 51-64. (3) Wales, T. E.; Engen, J. R. Mass Spectrom. Rev. 2006, 25, 158-170. (4) Smith, D. L.; Deng, Y.; Zhang, Z. J. Mass Spectrom. 1997, 32, 135-146. (5) Mazon, H.; Marcillat, O.; Forest, E.; Smith, D. L.; Vial, C. Biochemistry 2004, 43, 5045-5054. (6) Chik, J. K.; Vande Graaf, J. L.; Schriemer, D. C. Anal. Chem. 2006, 78, 207-214. (7) Cravello, L.; Lascoux, D.; Forest, E. Rapid Commun. Mass Spectrom. 2003, 17, 2387-2393. (8) Katta, V.; Chait, B. T. J. Am. Chem. Soc. 1993, 115, 6317-6321. (9) Kaltashov, I. A.; Eyles, S. J. J. Mass Spectrom. 2002, 37, 557-565. (10) Hoerner, J. K.; Xiao, H.; Dobo, A.; Kaltashov, I. A. J. Am. Chem. Soc. 2004, 126, 7709-7717.

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digestion experiments.11,12 A conceptual problem with these approaches is the widely accepted notion that peptide bond CID involves mobile protons. Theoretical and experimental studies strongly suggest that fragmentation should be accompanied by extensive intramolecular H/D migration (“scrambling”).13-17 Such an effect would lead to a partial or complete randomization of the spatial HDX pattern, such that site-specific information is lost. This potential problem notwithstanding, several research groups have attempted to use CID for analyzing proteins after deuterium labeling. For example, Anderegg et al.18 reported the successful application of triple-quadrupole MS/MS for sitespecific HDX experiments. Akashi et al.19-21 and Kaltashov et al.10,22,23 employed nozzle-skimmer CID FT-ICR-MS with relatively little apparent scrambling. Using quadrupole ion trap MS/MS studies, the laboratories of Smith11 and Deinzer12 found that the labeling patterns of b ions were consistent with previous NMR data, whereas y′′ ions did not provide reliable information. Various degrees of scrambling in Q-TOF MS/MS were reported by Heck et al.,24,25 who also suggested that peptides cationized with alkali metal ions are more resistant to intramolecular H/D migration than their protonated counterparts. They argued that metal-bound gas-phase peptides likely adopt more compact conformations, which might restrict the extent of scrambling.24,25 Complete scrambling for both b and y′′ ions was found by Jorgensen and co-workers26 in MS/MS experiments on a Q-TOF instrument and by Johnson and co-workers14 for measurements employing triplequadrupole MS. Virtually complete H/D scrambling was also observed in studies employing SORI-CID.10,27 Unfortunately, the partially conflicting findings of the studies described above do not result in a consistent picture regarding the occurrence of scrambling (or the lack thereof) for a given set of conditions. The aim of the current work is to help clarify the feasibility of HDX-CID MS experiments. Q-TOF MS/MS was used in an attempt to monitor spatially resolved deuteration levels under very specific conditions. We employed extremely short HDX times in order to mimic the exchange regime typically encountered in kinetic pulse-labeling studies for the detection of short-lived (11) Deng, Y.; Pan, H.; Smith, D. L. J. Am. Chem. Soc. 1999, 121, 1966-1967. (12) Kim, M.-Y.; Maier, C. S.; Reed, D. J.; Deinzer, M. L. J. Am. Chem. Soc. 2001, 123, 9860-9866. (13) Paizs, B.; Suhai, S. Mass Spectrom. Rev. 2005, 24, 508-548. (14) Johnson, R. S.; Krylov, D.; Walsh, K. A. J. Mass Spectrom. 1995, 30, 386387. (15) Harrison, A. G.; Yalcin, T. Int. J. Mass Spectrom. 1997, 165/166, 339-347. (16) Tang, X.-J.; Thibault, P.; Boyd, R. K. Anal. Chem. 1993, 65, 2824-2834. (17) Aribi, H.; Orlova, G.; Hopkinson, A. C.; Siu, M. K. W. J. Phys. Chem. A 2004, 108, 3844-3853. (18) Anderegg, R. J.; Wagner, D. S.; Stevenson, C. L.; Borchardt, R. T. J. Am. Soc. Mass Spectrom. 1994, 5, 425-433. (19) Akashi, S.; Naito, Y.; Takio, K. J. Mol. Biol. 1999, 71, 4974-4980. (20) Akashi, S.; Takio, K. Protein Sci. 2000, 9, 2497-2505. (21) Akashi, S.; Takio, K. J. Am. Soc. Mass Spectrom. 2001, 12, 1247-1253. (22) Eyles, S. J.; Speir, J. P.; Kruppa, G. H.; Gierasch, L. M.; Kaltashov, I. A. J. Am. Chem. Soc. 2000, 122, 495-500. (23) Hoerner, J. K.; Xiao, H.; Kaltashov, I. A. Biochemistry 2005, 44, 1128611294. (24) Demmers, J. A. A.; Haverkamp, J.; Heck, A. J. R.; Koeppe, R. E.; Killian, A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 3189-3194. (25) Demmers, J. A. A.; Rijkers, D. T. S.; Haverkamp, J.; Killian, J. A.; Heck, A. J. R. J. Am. Chem. Soc. 2002, 124, 11191-11198. (26) Jorgensen, T. J. D.; Gardsvoll, H.; Ploug, M.; Roepstorff, P. J. Am. Chem. Soc. 2005, 127, 2785-2793. (27) McLafferty, F. W.; Guan, Z.; Haupts, U.; Wood, T. D.; Kelleher, N. L. J. Am. Chem. Soc. 1998, 120, 4732-4740.

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protein folding intermediates.28-34 These experiments use a slightly basic pD to ensure that the exchange of unprotected hydrogens occurs on a millisecond time scale.32,35 The availability of reliable MS/MS protocols for measuring site-specific deuteration levels in folding intermediates would be a significant advance, as it would allow valuable structural information on these highly elusive species to be obtained.31 The system investigated here is a 22-residue, zinc-binding domain (ZBD) derived from the C-terminus of Escherichia coli SecA, an ATPase involved in protein translocation.36 Metalation induces the formation of a well-defined three-dimensional structure, where the tetracoordinate Zn2+ is ligated by the side chains of Cys8, Cys10, Cys19, and His20 (Figure 1).37 Zn2+-bound ZBD (Zn-ZBD) was chosen as a model system for this study because it has several properties that we presumed would render it particularly resistant to intramolecular H/D migration. First, the structural rigidity conferred by metal binding should reduce the extent of scrambling.24,25 Second, ZBD is relatively basic (pI ≈ 10), such that mobile charge carriers might be sequestered on sites with high proton affinity (2 Arg, 1 His, 4 Lys, the N-terminal Lys being acetylated).38-40 Third, an extensive network of Hbonds37 should confer exchange protection, and this would form the basis upon which we would measure site-specific HDX labeling patterns. We find that Q-TOF MS/MS on pulse-labeled Zn-ZBD produces fragment ions that exhibit a very distinct deuteration pattern. At first glance, this might indicate that site-selective information is preserved during fragmentation. However, the measured HDX levels are not correlated with those expected based on the known Zn-ZBD structure, indicating that significant scrambling does in fact take place. The observation of similar fragment deuteration patterns under different labeling conditions strongly suggests that scrambling is not the only conceptual problem that has to be taken into account for the interpretation of HDX-CID data. EXPERIMENTAL SECTION Chemicals. ZBD (Ac-KVGRNDPCPCGSGKKYKQCHGR, neutral monoisotopic mass of the apoform 2459.17 Da) was obtained by solid-phase FMOC synthesis, purified by reversed-phase HPLC, (28) Miranker, A.; Robinson, C. V.; Radford, S. E.; Aplin, R.; Dobson, C. M. Science 1993, 262, 896-900. (29) Tsui, V.; Garcia, C.; Cavagnero, S.; Siuzdak, G.; Dyson, H. J.; Wright, P. E. Protein Sci. 1999, 8, 45-49. (30) Heidary, D. K.; Gross, L. A.; Roy, M.; Jennings, P. A. Nat. Struct. Biol. 1997, 4, 725-731. (31) Konermann, L.; Simmons, D. A. Mass Spectrom. Rev. 2003, 22, 1-26. (32) Simmons, D. A.; Dunn, S. D.; Konermann, L. Biochemistry 2003, 42, 58965905. (33) Pan, J. X.; Wilson, D. J.; Konermann, L. Biochemistry 2005, 44, 8627-8633. (34) Hossain, B. M.; Konermann, L. Anal. Chem. 2006, 78, 1613-1619. (35) Bai, Y.; Milne, J. S.; Mayne, L.; Englander, S. W. Proteins: Struct. Funct. Genet. 1993, 17, 75-86. (36) Fekkes, P.; de Wit, J. G.; Boorsma, A.; Friesen, R. H. E.; Driessen, A. J. M. Biochemistry 1999, 38, 5111-5116. (37) Dempsey, B. R.; Wrona, M.; Moulin, J. M.; Gloor, G. B.; Jalilehvand, F.; Lajoie, G. A.; Shaw, G. S.; Shilton, B. H. Biochemistry 2004, 43, 93619371. (38) Kapp, E. A.; Schutz, F.; Reid, G. E.; Eddes, J. S.; Moritz, R. L.; O’Hair, R. A. J.; Speed, T. P.; Simpson, R. J. Anal. Chem. 2003, 75, 6251-6264. (39) Martin, D. B.; Eng, J. K.; Nesvizhskii, A. I.; Gemmill, A.; Aebersold, R. Anal. Chem. 2005, 77, 4870-4882. (40) Gu, C.; Tsaprailis, G.; Breci, L.; Wysocki, V. H. Anal. Chem. 2000, 72, 58045813.

interface. N2 desolvation gas was used at a flow rate of 500 L h-1 and a temperature of 80 °C. The labeling time was controlled by adjusting the distance between mixing point and capillary exit.41 Time points between 25 and 750 ms were studied using the capillary setup; manual mixing was employed for longer times (hours to days). Zn-ZBD MS/MS experiments were carried out on a Q-TOF Ultima (Micromass/Waters) operated in W-reflectron mode at a resolution of 15 000 fwhm. The drastically lower sensitivity under these conditions (compared to standard Vreflectron mode) necessitated total data acquisition times of 2-4 h in MS/MS mode. Fragmentation experiments were performed on the 3+ charge state of Zn-ZBD. Slight detuning of the quadrupole was necessary to ensure transmission of the entire isotope envelope of the labeled precursor ion into the Ar-filled collision cell. An MS/MS collision voltage of 33 V resulted in the most intense fragment ion signals. V-Reflectron mode at a resolution of 10 000 fwhm was used for MS/MS control experiments on bradykinin. All experiments were carried out at room temperature 22 ( 1 °C, unless noted otherwise. Data Analysis. The isotope distribution D for each ion after labeling was modeled as a convolution,

D ) N*B

(1)

where N is that natural isotope distribution (ProteinProspector, UCSF). The binomial distribution B is given by

B(p,n,k) ) Figure 1. NMR structure of the zinc-binding domain (Zn-ZBD) studied in this work. The figure was rendered using PyMOL based on PBD file 1SX1, model 1.37 (A) All-atom representation of Zn-ZBD; C (green), H (white), N (blue), O (red), and S (yellow). (B) Zn2+coordinating residues (note that the orientation of the molecule is different from that in panel A).

and lyophilized. Samples prepared for ESI-MS were dissolved in water to a final concentration of 25 µM along with 5 µM bradykinin (RPPGFSPFR) as internal standard, obtained from Bachem (Bubendorf, Switzerland), and ultrapure 65 µM ZnCl2. The pH was adjusted to 9.0 using dilute ammonium hydroxide solution. The labeling buffer used consisted of deuterium oxide (99.9%) from Cambridge Isotope Laboratories (Andover, MA), adjusted to pD 9.0 using ammonium hydroxide, 99 atom % D (Aldrich, St. Louis, MO). On-Line HDX ESI-MS and MS/MS. Isotope labeling experiments were performed using an on-line, continuous-flow capillary mixing system similar to that employed for previous studies in our laboratory.41 The mixer was assembled within a modified Z-spray ESI source. Zn-ZBD solution and D2O labeling buffer were pumped into the mixing device at flow rates of 10 and 40 µL min-1, respectively, using a Harvard model 22 syringe pump (South Nattick, MA). The resulting Zn-ZBD concentration after mixing was 5 µM at a nominal D2O content of 80%. This solution was electrosprayed directly (i.e., without using a makeup solvent or LC) at a capillary voltage of 3 kV. The cone voltage was kept low (10 V) in order to avoid collisional activation in the ion sampling (41) Wilson, D. J.; Konermann, L. Anal. Chem. 2003, 75, 6408-6414.

n! pk(1 - p)n-k k!(n - k)!

(2)

In this equation, n is the total number of exchangeable hydrogens in the ion of interest and k ) 0, 1, ..., n. The deuteration level p (0 e p e 100%) of each ion was determined from the simulated distribution D that resulted in the best fit to the experimental data. The quality of the fits was determined by visual inspection, placing the highest emphasis on the most intense peaks of the isotope distribution. Error estimates for each ion were performed by successively lowering (or increasing) the value of p until unacceptable deviations between the modeled distribution and the experimental spectrum were obtained, as illustrated in Figure 2. The different error bars reported for the various ions reflect the different signal-to-noise ratios of individual ionic signals. Protected Hydrogens in ZBD. The NMR structure of ZnZBD (PDB code 1SX1)37 was used to determine which exchangeable sites are most likely involved in stable H-bonds and would thus be expected to exchange with the slowest rates. H-bond donors and acceptors were identified by CNS42 and subsequently verified by visual inspection using the program O.43 Criteria for a good H-bond were that the distance between the donor and acceptor atoms was 3.5 Å or less and that the angle between the donor, acceptor, and acceptor carbon was ∼120°. As a result of this analysis, 11 amide backbone NsH.....OdC H-bonds were (42) Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros, P.; GrosseKunstleve, R. W.; Jiang, J.-S.; Kuszewski, J.; Nilges, M.; Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L. Acta Crystallogr., D: Biol. Crystallogr. 1998, 54, 905-921. (43) Jones, T. A.; Bergdoll, M.; Kjeldgaard, M. In Crystallographic and Modeling Methods in Molecular Design; Bugg, C. E., Ealick, S. E., Eds.; Springer: New York, 1990; pp 189-195.

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Figure 2. Determination of the deuteration level (parameter p in eq 2) and its corresponding error illustrated for b14+ after 200 ms of isotope labeling. (A) Experimental spectrum and modeled distribution that best describes the experimental data (deuteration level 62%, filled circles). (B) Experimental data superimposed on distributions calculated for deuteration levels of 59 (filled squares) and 65% (open circles). Evidently, the HDX levels of the modeled distributions in (B) do not match the experimental data; one is too high the other one is too low. Based on this analysis the deuteration level b14+ is reported as 62 ( 3%. Table 1. H-bonds in Zn-ZBD, Based on the NMR Structure 1SX137 H-bond acceptora

H-bond donorb

distance (Å)

angle (deg)

Val2 Gly3 Lys14 Cys8 Cys10 S (side chain) Cys10 S or Cys19 S (side chain)c Ser12 O (side chain) Asp6 or Arg4c Lys15 Lys15 or Tyr16c Tyr16 Tyr16 or Cys19c

Arg4 Asp6 Cys8 Gly11 Ser12 Ser12 -OH (side chain) Lys14 Tyr16 Gln18 Cys19 Gly21 Arg22

2.8 2.5 2.7 3.1 3.4 3.3 or 3.6

103 147 137 128 116 163 or 104

3.5 3.2 or 3.2 2.8 2.6 or 2.7 2.6 2.8 or 3.2

131 137 or 149 118 165 or 120 114 127 or 118

a Refers to backbone CdO, unless noted otherwise. b Refers to backbone NsH, unless noted otherwise. c Either acceptor can interact with the donor.

identified (Table 1). The only good side-chain donor was Ser12, which can donate a H-bond to the Zn-ligating thiolates of either Cys10 or Cys19. Thus, a total of 12 hydrogens in Zn-ZBD should exhibit slow exchange. RESULTS AND DISCUSSION The ESI mass spectrum of Zn-ZBD is dominated by a triply charged species. Considering that three of the four Zn2+ ligands are deprotonated thiolates, these ions may be formally interpreted as [Zn‚ZBD- + 4H+]3+ (Figure 3). When analyzing Zn-ZBD in aqueous solution, the natural isotope distribution of zinc gives the peak intensity progression a somewhat “irregular” appearance (Figure 4A). Triply charged Zn-ZBD possesses a total of 49 exchangeable hydrogens, including contributions from the amide backbone, side chains, termini, and the charge carriers. Twelve of these are 156 Analytical Chemistry, Vol. 79, No. 1, January 1, 2007

Figure 3. Cartoon representation of the zinc coordination in ZnZBD based on PBD file 1SX1.37 Residues marked with an asterisk correspond to the H-bond donors in Table 1. The diagram is not meant to imply any stereochemical information.

expected to undergo slow exchange due to H-bonding (Table 1). The deuteration level of Zn-ZBD was probed at various time points after exposure to 80% D2O in the presence of bradykinin as internal standard. All 19 labile hydrogens in bradykinin undergo exchange within a few milliseconds under the slightly basic conditions used here.8,44 Thus, virtually complete labeling of the internal standard was observed for all time points studied. As an example, Figure 4B (inset) shows the mass distribution of bradykinin after 200 ms of labeling, resulting in a deuteration level of 78.1 ( 0.5%. The minor discrepancy from the expected 80% value may be caused by slight deviations of the nominal 20:80 mixing ratio delivered by the syringe pumps or by a small degree of back exchange in the ion source.14,18,19 The deuteration level of Zn-ZBD for t ) 200 ms is 65.8 ( 0.5%, i.e., much lower than for bradykinin under the same conditions (Figure 4B). This difference reflects the fact that Zn-ZBD possesses protected sites, whereas bradykinin does not.8,44 Figure 4C depicts the isotope distribution of Zn-ZBD after 4 days of incubation in the labeling buffer. HDX under these conditions has long gone to completion, and the deuteration levels measured for both Zn-ZBD and bradykinin are 81.0 ( 0.5%. Figure 5 shows the measured deuteration kinetics of Zn-ZBD, normalized to account for the 20% H2O content of the labeling buffer. The experimental data points are well described by a singleexponential fit. The extrapolated burst phase amplitude of the fitted curve corresponds to 38 hydrogens that are labeled within the dead time of the experiment. A total of ∼11 sites undergo HDX much more slowly (keff ) 1.6 s-1). Taking into account the uncertainty of the extrapolated kinetic amplitudes, the observed 11:38 ratio of slowly to rapidly exchanging sites is in acceptable agreement with the ratio of 12:37 predicted based on the NMR structure of Zn-ZBD (Table 1). Before proceeding to MS/MS studies with isotope labeling, we explored the fragmentation behavior of nondeuterated Zn-ZBD. Selective CID of the triply charged precursor ion (depicted in Figure 4A) provides extensive sequence coverage by both b and y′′ ions. However, the relative abundance of the various fragmen(44) Hossain, B. M.; Simmons, D. A.; Konermann, L. Can. J. Chem. 2005, 83, 1953-1960.

Figure 4. ESI mass spectra showing the 3+ charge state of ZnZBD. (A) Aqueous solution, no isotope labeling; (B) after 200 ms of HDX; (C) after 4 days of HDX. Filled circles represent simulated isotope distributions for deuteration levels of (A) zero, (B) 65.8 ( 0.5%, and (C) 81.0 ( 0.5%. Panel B, inset: isotope distribution of the doubly charged bradykinin internal standard after 200 ms of labeling, deuteration level 78.1 ( 0.5%.

It appears as a doubly charged species that retains zinc (Zn-y16′′2+, data not shown). The only other metalated fragment is Zn-b202+ (Figure 6B). It is interesting to note that both Zn-b202+ and Zny16′′2+ contain the Cys8...His20 segment carrying all four residues that are involved in zinc ligation (Figure 3). y14′′+ provides an example of a zinc-free ion that originates from fragmentation within this Cys8...His20 segment (Figure 6D). The key question investigated in this study is whether CID MS/MS on a Q-TOF is a viable means to pinpoint the locations of slowly exchanging hydrogens in Zn-ZBD. We arbitrarily chose to focus our attention on the t ) 200 ms labeling time point. Triply charged Zn-ZBD with an overall deuteration level of 65.8 ( 0.5% served as precursor species in these fragmentation experiments (Figure 4B). A total of 11 b and 15 y′′ ions exhibited signal-tonoise ratios that were adequate for a reliable analysis. Representative examples of these spectral data are provided in Figure 6EH, and a summary of all measured deuteration levels is given in Figure 7A, B (filled blue circles with error bars). Also shown is the 65.8% deuteration level of the precursor ion (black dotted lines). Based on the H-bonding pattern of Zn-ZBD (Table 1), it is possible to predict the deuteration levels that would be expected for b and y′′ ions in the absence of scrambling. For calculating these “no-scrambling-profiles”, it was supposed that all unprotected sites, including the two extra charge carriers on y′′ ions,19 were fully exchanged to 80%. The observation of single-exponential HDX (Figure 5) implies that the individual exchange rate constants of all 12 protected H-bond donors are very similar.45 Thus, it can be assumed that these sites are deuterated to the same extent for t ) 200 ms. From the 65.8% HDX level of intact Zn-ZBD, it follows that these protected sites exhibit an average deuteration level of 0.275 × 80% ) 22%. Thus, the expected no-scrambling HDX level, pexpect, for every fragment ion was calculated according to

pexpect )

Figure 5. HDX kinetics of Zn-ZBD. The data have been normalized to account for the presence of 20% H2O in the labeling buffer. The solid line is an exponential fit of the form y0 + y1(1 - exp[-kefft]), with y0 ) 38 (burst amplitude, indicated by the bold arrow), y1 ) 11, and keff ) 1.6 s-1.

tation products encompasses more than 2 orders of magnitude, and some of the weaker signals could not be used for the subsequent analyses due to signal-to-noise limitations. Low molecular weight product ions generally resulted in higher signal intensities than larger fragments. The most abundant product ion is b6+ (Figure 6A), which is to be expected based on the known labile nature of Asp-Pro bonds during CID,13,38,40 in this case, Asp6-Pro7. The complementary y′′ ion has a much lower intensity.

(nunprot + nprot × 0.275) × 80% n

(3)

where nunprot and nprot are the numbers of unprotected and protected sites, respectively, with (nunprot + nprot) ) n. For example, the first three fragments of the b ion series do not contain any protected sites, corresponding to pexpect values of 80%. y1′′ contains a total of eight exchangeable sites, one of which is protected, resulting in pexpect ) 72.8%. A value of 67.1% is predicted for y2′′ (9 exchangeable sites in total, 2 protected). The no-scrambling profiles calculated in this way are depicted as red solid lines in Figure 7A, B. Inspection of the data summarized in Figure 7A, B reveals that the experimentally determined labeling levels of b and y′′ ions bear no resemblance to the expected no-scrambling-profiles. The most striking deviations are observed in the range of b2-b8 (Figure 7A), and y1′′-y9′′ (Figure 7B). These observations have several implications. Zn-ZBD was selected as a model system for this study because it was thought that metal binding, as well as its basic character, would render this protein domain highly resistant to intramolecular H/D migration during CID.24,25,38-40 Nonetheless, the deuteration levels measured for pulse-labeled (45) Hvidt, A.; Nielsen, S. O. Adv. Protein Chem. 1966, 21, 287-386.

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Figure 6. Examples of b and y′′ fragment ions generated by CID MS/MS of Zn-ZBD on a Q-TOF instrument. The deuteration levels shown in each panel were determined by fitting simulated distributions (filled circles) to the mass envelopes. Top row (A-D), natural isotope abundance (no labeling); middle row (E-H), labeling time 200 ms; bottom row (I-L), labeling time 4 days. The inset in panel B shows an expansion of the unlabeled Zn-b202+ signal, which overlaps with an unidentified singly charged ion.

Zn-ZBD not seem to retain any site-selective HDX information. Some earlier studies had suggested that spatially resolved deuteration levels can be extracted from b fragments, but not from y′′ ions.11,12 In the case of Zn-ZBD, this result cannot be confirmed, since both types of ions fail to preserve any site-selective information. We conclude that CID MS/MS is not a viable approach for determining spatially resolved deuteration levels in Zn-ZBD, a result that is consistent with previous data on a number of other systems.14,26,27 It is an intriguing finding that the deuteration levels measured after pulse-labeling (Figure 7A, B) strongly diverge from the expected no-scrambling-profiles and from the uniform distributions that might be expected for complete scrambling. In the absence of any prior information, the measured deuteration pattern could erroneously be ascribed to HDX protection. For example, “apparent protection” might be deduced from the very low labeling level of b2 (Figure 7A), which is in complete disagreement with the NMR structure of Zn-ZBD. It is interesting to compare these MS/MS data for t ) 200 ms with measurements on samples that had been exposed to the labeling buffer for 4 days (Figure 6I-L, Figure 7C, D). As discussed above, this treatment results in a precursor deuteration level of 81.0 ( 0.5% (Figure 4C). The rationale behind this control experiment was that under these conditions every exchangeable site in solution will be labeled to a degree that is solely determined by the isotopic composition of the solvent. In other words, all of the b and y′′ fragments generated from uniformly labeled Zn-ZBD would be expected to show a deuteration level of 81.0 ( 0.5%, a 158 Analytical Chemistry, Vol. 79, No. 1, January 1, 2007

situation that mimics complete scrambling. Remarkably, the fragments do not exhibit a uniform HDX behavior under these conditions (Figure 7C, D). Instead, the deuteration patterns for b and y′′ ions strongly resemble those obtained for a labeling time of 200 ms (compare panels A/C, and B/D in Figure 7). To completely eliminate residual HDX protection as possible cause of these deuteration patterns, the control experiments were repeated with an extended labeling period of 11 days at an elevated temperature of 40 °C. The resulting deuteration levels were virtually identical to those depicted in Figure 7C and D (data not shown). The fact that the deuteration pattern for t ) 200 ms resembles that of uniformly labeled Zn-ZBD provides evidence that complete scrambling during CID does in fact take place under the conditions used here. Additionally, however, other factors must play a role, otherwise the HDX levels of all fragments in Figure 7 would coincide with those of their corresponding precursor ions. It is an intriguing question whether CID fragments exhibiting different deuteration levels can also be generated from other uniformly labeled peptides or whether this behavior is specific to Zn-ZBD. MS/MS experiments were carried out on uniformly labeled bradykinin, following incubation in 80% D2O for 30 h at pH 9.0. Subsequent mass analysis confirmed complete labeling to a level of 80.0 ( 0.5% (Figure 8A). Fragmentation of the doubly charged precursor under these conditions results in several product ions having deuteration levels close to 80% (e.g., b5, Figure 8B). Other fragments, however, exhibit labeling levels that differ

Figure 7. Deuteration levels of b and y′′ ions for labeling times of 200 ms (A, B) and 4 days (C, D). Red solid lines in (A, B) denote profiles expected in the absence of scrambling. Dotted black lines indicate the deuteration level of the precursor ion, i.e., 65.8 ( 0.5% for (A, B) and 81.0 ( 0.5% for (C, D). Blue filled circles with error bars represent values determined experimentally. The data for both b and y′′ ions are aligned with the ZBD sequence shown in N-terminal f C-terminal direction at the top of panels A and B. Data points shown as “b22” and “y22” correspond to the intact precursor ion, depicted in Figure 4.

considerably from that of the precursor. As an example, Figure 8C shows that the data measured for y4′′ clearly do not match a modeled 80% distribution. A more satisfactory fit is obtained for a labeling level of 74.5% (Figure 8D). The data for all b and y′′ ion are summarized in Figure 9, illustrating that CID fragments generated from uniformly labeled bradykinin can have deuteration levels significantly above (b7, b8) or below (b2, y1′′, y3′′, y4′′) that of the precursor ion. Thus, the occurrence of nonuniform fragment deuteration is not limited to Zn-ZBD; instead, this phenomenon is also seen for other peptides. The physical reasons underlying the nonuniform deuteration patterns cannot be uncovered with certainty from the experimental data presented here. Nonetheless, we will briefly speculate on some factors that may be relevant. HDX-CID studies generally rely on the implicit assumption that the fragmentation behavior of a peptide is not affected by its deuteration level. The observations made here suggest that this assumption may not always be justified. It is well known that the stability of individual amide bonds under CID conditions depends on sequence, conformation, and charge state.38,46 Possibly, the stability can additionally be modulated by kinetic isotope effects related to differences in the amide deuteration status.47-49 The following thought experiment illustrates the consequences of such a scenario: First consider a

perfectly scrambled peptide and assume that the fragmentation efficiency of a particular amide bond is slightly higher in its -CONH- form. Thus, the resulting fragment ions will be preferentially formed from the low-mass portion of the precursor H/D isotope envelope (keeping in mind that this envelope includes a wide range of deuteration states k, according to eq 2). Similarly, the fragmentation efficiency of another amide bond in the same peptide may be higher in its -CO-ND- form, thereby slightly favoring the formation of fragments from precursor ions carrying more deuterons. In this hypothetical scenario, individual fragments of a completely scrambled peptide can exhibit deuteration levels that are different from each other and that deviate from that of the precursor ion. Other factors could play role as well. For example, a proton enrichment in C-terminal Arg residues might occur if the proton affinity of these residues is larger than their deuteron affinity. Such a mechanism might contribute to the relatively low y1′′ HDX levels of both Zn-ZBD and bradykinin (Figure 7B, D, Figure 9B). There is some evidence that spatial H/D partitioning effects of this kind can occur in solution;50,51 however, the relevance of those previous studies for the behavior of peptides in the gas phase is not clear. Certainly, the validity of these suggested scenarios has to be explored in future studies.

(46) Huang, Y.; Triscari, J. M.; Tseng, G. C.; Pasa-Tolic, L.; Lipton, M. S.; Smith, R. D.; Wysocki, V. H. Anal. Chem. 2005, 77, 5800-5813. (47) Mirza, S. P.; Krishna, P.; Prabhakar, S.; Vairamani, M.; Giblin, D.; Gross, M. L. Int. J. Mass Spectrom. 2003, 230, 175-183. (48) Herrmann, K. A.; Kuppannan, K.; Wysocki, V. H. Int. J. Mass Spectrom. 2006, 249-250, 93-105. (49) Derrick, P. J.; Donchi, K. F. In Comprehensive Chemical Kinetics; Bamford, C. H., Tipper, C. F. H., Eds.; Elsevier: Amsterdam, 1983; Vol. 24.

(50) Bowers, P. M.; Klevit, R. E. Nat. Struct. Biol. 1996, 3, 522-531. (51) Shi, Z.; Krantz, B. A.; Kallenbach, N.; Sosnick, T. R. Biochemistry 2002, 41, 2120-2129. (52) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, 3265-3266. (53) Kweon, H. K.; Hakansson, K. Analyst 2006, 131, 275-280. (54) Charlebois, J. P.; Patrie, S. M.; Kelleher, N. L. Anal. Chem. 2003, 75, 32633266.

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Figure 9. Deuteration levels of b (A) and y′′ (B) ions obtained by fragmentation of uniformly labeled bradykinin. Data points shown as “b9” and “y9” correspond to the intact precursor ion, depicted in Figure 8A. Dotted lines indicate the 80.0 ( 0.5% deuteration level of the precursor ion.

Figure 8. (A) Isotope distribution of doubly charged bradykinin under uniform labeling conditions. The resulting CID fragments, b5 and y4′′ are shown in (B) and (C), respectively. Also shown in (A-C) are modeled isotope envelopes for a deuteration level of 80% (filled circles). Note that the modeled distribution in (C) does not match the experimental data. Panel D shows the same experimental data as (C), but with a distribution modeled for 74.5% deuteration, which results in a better fit.

CONCLUSIONS The use of gas-phase fragmentation strategies for spatially resolved HDX studies holds potential promise for a wide range of applications, as evidenced by the activity of numerous research groups in this area. Unfortunately, the current work contributes to the growing body of evidence that CID methods are not a generally viable tool for monitoring HDX in site-specific experiments. Using MS/MS on a Q-TOF instrument, we found that the deuteration patterns of both b and y′′ ions do not at all match the profiles that would be expected based on the known Zn-ZBD

160 Analytical Chemistry, Vol. 79, No. 1, January 1, 2007

structure. It appears that H/D scrambling is chiefly responsible for this effect. In addition, however, our results point to other issues that have to be taken into account as well. Unexpectedly, even uniformly labeled Zn-ZBD and bradykinin (which should mimic perfect scrambling) do not exhibit constant HDX levels in their various CID fragments. This behavior may be caused by kinetic isotope effects that modulate the fragmentation efficiency of peptide bonds during CID, but other factors such as spatial H/D partitioning could play a role as well. Such “nonideal” behavior can give rise to a misinterpretation of data, particularly in cases where structural information on the system under investigation is not available. Studies aiming to determine sitespecific deuteration levels by gas-phase fragmentation methods should always include control experiments on uniformly labeled samples to explore the occurrence of such fragmentation artifacts. It is hoped that ion activation methods other than CID, such as electron capture dissociation,52-54 will provide a more robust approach for spatially resolved HDX-MS studies. ACKNOWLEDGMENT We thank Drs. K. W. M Siu and M. J. Stillman for helpful discussions. This work was financially supported by the Natural Sciences and Engineering Research Council of Canada, the Canada Foundation for Innovation, the Ontario Research and Development Challenge Fund, and the Canada Research Chairs Program.

Received for review July 12, 2006. Accepted October 10, 2006. AC061261F