Gas-Phase Fragmentation of Peptides by MALDI in-Source Decay with

Jul 22, 2008 - To achieve a fundamental understanding of the function of proteins and protein complexes at the molecular level, it is crucial to obtai...
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Anal. Chem. 2008, 80, 6431–6435

Gas-Phase Fragmentation of Peptides by MALDI in-Source Decay with Limited Amide Hydrogen (1H/2H) Scrambling Nicolai Bache, Kasper D. Rand, Peter Roepstorff, and Thomas J. D. Jørgensen* Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark To achieve a fundamental understanding of the function of proteins and protein complexes at the molecular level, it is crucial to obtain a detailed knowledge about their dynamic and structural properties. The kinetics of backbone amide hydrogen exchange is intimately linked to the structural dynamics of the protein, and in recent years, the monitoring of the isotopic exchange of these hydrogens by mass spectrometry has become a recognized method. At present, the resolution of this method is, however, limited and single-residue resolution is typically only obtained for a few residues in a protein. It would therefore be desirable if gas-phase fragmentation could be used to localize incorporated deuterons as this would ultimately lead to single-residue resolution. A central obstacle for this approach is, however, the occurrence of intramolecular migration of amide hydrogens upon activation of the gaseous protein (i.e., hydrogen scrambling). Here we investigate the occurrence of scrambling in selectively labeled peptides upon fragmentation by matrix-assisted laser desorption/ionization in-source decay (MALDI ISD). We have utilized peptides with a unique regioselective deuterium incorporation that allows us to accurately determine the extent of scrambling upon fragmentation. Our results show that the level of scrambling upon MALDI ISD is so low that the solution deuteration pattern is readily apparent in the gas-phase fragment ions. These results suggest that MALDI ISD may prove useful for hydrogen exchange studies of purified peptides and small proteins. Protein function (and dysfunction) is intimately linked to the dynamic nature of protein structure.1 A well-known example is enzymatic activity and its allosteric regulation, which involve an ensemble of interconverting conformations.2 Amide hydrogen (1H/2H) exchange combined with mass spectrometry has become a recognized method for the study of protein structural dynamics.3,4 With this method, a global exchange experiment is typically initiated by diluting a given protein into deuterated buffer. The * To whom correspondence should be addressed. Telephone: +45 6550 2414. Fax: +45 6550 2467. E-mail: [email protected]. (1) Henzler-Wildman, K.; Kern, D. Nature 2007, 450, 964–972. (2) Hammes, G. G. Biochemistry 2002, 41, 8221–8228. (3) Wales, T. E.; Engen, J. R. Mass Spectrom. Rev. 2006, 25, 158–170. (4) Rand, K. D.; Jørgensen, T. J. D.; Olsen, O. H.; Persson, E.; Jensen, O. N.; Stennicke, H. R.; Andersen, M. D. J. Biol. Chem. 2006, 281, 23018–23024. 10.1021/ac800902a CCC: $40.75  2008 American Chemical Society Published on Web 07/22/2008

global exchange kinetics is readily obtained by monitoring the mass increase of the intact protein (due to its deuterium uptake) as a function of exchange time. To obtain information about local deuterium incorporation, the labeled protein is proteolytically cleaved with pepsin under conditions where the isotopic exchange reaction is quenched (i.e., low temperature and low pH). The labeled peptic peptides are typically separated by reversed-phase chromatography (under quench conditions and using a short gradient) and subsequently analyzed by electrospray ionization mass spectrometry.5 Alternatively, MALDI TOF mass spectrometry can be used to analyze the labeled peptic peptides without prior chromatographic separation.6 The resolution of these approaches is, however, limited by the number and sizes of peptic peptides, and single-residue resolution is generally only obtained for a few residues. Consequently, much effort is currently devoted to find a gas-phase fragmentation method that allows the solution deuteration pattern to be deduced from mass spectrometry experiments as this would ultimately lead to desired single-residue resolution. The occurrence of hydrogen scrambling is, however, the main hindrance for achieving this goal. Presently, low-energy collision-induced dissociation (CID) is the most widespread technique for gas-phase fragmentation of peptides. Unfortunately, CID results in extensive hydrogen scrambling in protonated peptides and proteins.7–15 We have recently developed peptide probes that accurately measure the occurrence of intramolecular (5) Engen, J. R. ; Smith, D. L. In Mass Spectrometry of Proteins and Peptides: Mass Spectrometry of Proteins and Peptides; Chapman, J. R., Ed.; Humana Press: Totowa, NJ, 2000; Vol. 146, pp 95-112. (6) Mandell, J. G.; Falick, A. M.; Komives, E. A. Anal. Chem. 1998, 70, 3987– 3995. (7) 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. (8) Ferguson, P. L.; Pan, J. X.; Wilson, D. J.; Dempsey, B.; Lajoie, G.; Shilton, B.; Konermann, L. Anal. Chem. 2007, 79, 153–160. (9) Harrison, A. G.; Yalcin, T. Int. J. Mass Spectrom. 1997, 165, 339–347. (10) Johnson, R. S.; Krylov, D.; Walsh, K. A. J. Mass Spectrom. 1995, 30, 386– 387. (11) Jørgensen, T. J. D.; Bache, N.; Roepstorff, P.; Gårdsvoll, H.; Ploug, M. Mol. Cell. Proteomics 2005, 4, 1910–1919. (12) Jørgensen, T. J. D.; Gårdsvoll, H.; Ploug, M.; Roepstorff, P. J. Am. Chem. Soc. 2005, 127, 2785–2793. (13) McLafferty, F. W.; Guan, Z. Q.; Haupts, U.; Wood, T. D.; Kelleher, N. L. J. Am. Chem. Soc. 1998, 120, 4732–4740. (14) Mueller, D. R.; Eckersley, M.; Richter, W. J. Org. Mass Spectrom. 1988, 23, 217–222. (15) We note, however, that low levels of scrambling have been reported for in-source collisional activation of multiply protonated proteins: Hoerner, J. K.; Xiao, H.; Dobo, A.; Kaltashov, I. A. J. Am. Chem. Soc. 2004, 2126, 7709–7717.

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providing evidence for a limited vibrational excitation in the MALDI ISD process. These results show that the fragment ions retain their specific backbone labeling from solution and in particular c-ions and sodiated y-ions may be used to obtain sitespecific information about solution deuteration levels in peptides and small proteins. Furthermore, our results provide a fundamental insight into the ISD activation processes.

Figure 1. In-source decay of selectively labeled peptides by MALDI at elevated laser intensity. The peptides are deuterated at their amides in the C-terminal half (shown in blue), while the amides in the N-terminal half are protiated (shown in red). Measurement of the deuterium contents of the resulting N-terminal gas-phase fragments provides a sensitive gauge of the occurrence of scrambling during fragmentation.

amide hydrogen (1H/2H) migration (i.e., hydrogen scrambling) upon gas-phase activation.16 Furthermore, we have shown that electron capture dissociation (ECD) proceeds with a very low level of backbone amide hydrogen scrambling.17 As MALDI in-source decay (ISD) and ECD exhibit some common features (e.g., similar fragment ion types and preservation of labile post-translational modifications),18,19 we have undertaken this study to investigate the occurrence of hydrogen scrambling in selectively labeled peptides during MALDI ISD (Figure 1). In MALDI, the analytes are cocrystallized with a large molar excess of matrix molecules.20 The matrix in the present study is an aromatic acid, 2,5-dihydroxybenzoic acid (DHB), which has a strong absorption at the wavelength of the MALDI UV laser. When the laser pulse irradiates the sample, a plume of neutrals and ions is ejected from the surface. For peptide analytes, this process results in the formation of gaseous intact protonated peptides. When the laser intensity is increased somewhat above the threshold value for peptide ion production, the peptides fragment by a process termed in-source decay.21,22 In this process, the peptide backbone is cleaved at N-CR bonds yielding c- and z-type fragments. This fragmentation occurs on a very fast time scale as the fragment ions acquire the same kinetic energy as the noncleaved peptide ions during the acceleration in the MALDI ion source. The fragment ions are therefore well focused and resolved, and their m/z values are determined by the same calibration curve as the intact peptide ions.21 The fragmentation in ISD is thought to be initiated by a hydrogen radical transfer from the matrix to the protonated peptide,18,23 but the mechanistic details are largely unknown. In the present study, we have determined the extent of intramolecular migration of backbone amide hydrogens (i.e., scrambling) for various fragment ion types (c, z, y). A very low level of scrambling was generally observed, (16) Rand, K. D.; Jørgensen, T. J. D. Anal. Chem. 2007, 79, 8686–8693. (17) Rand, K. D.; Adams, C. M.; Zubarev, R. A.; Jørgensen, T. J. D. J. Am. Chem. Soc. 2008, 130, 1341–1349. (18) Kocher, T.; Engstrom, A.; Zubarev, R. A. Anal. Chem. 2005, 77, 172–177. (19) Lennon, J. J.; Walsh, K. A. Protein Sci. 1999, 8, 2487–2493. (20) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299–2301. (21) Brown, R. S.; Lennon, J. J. Anal. Chem. 1995, 67, 3990–3999. (22) Hardouin, J. Mass Spectrom. Rev. 2007, 26, 672–682. (23) Takayama, M. J. Am. Soc. Mass Spectrom. 2001, 12, 1044–1049.

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EXPERIMENTAL SECTION Materials. The synthetic peptides were obtained from Genscript Corp. (Piscataway, NJ). D2O (99.9%) was purchased from Cambridge Isotope Laboratories, (Andover, MA). The MALDI matrix, DHB, was obtained from Sigma-Aldrich (St. Louis, MO). All other chemicals were of the highest grade commercially available. Amide 1H/2H Exchange and Selective Labeling of Peptides. The synthetic peptides were fully deuterated by dissolution of lyophilized peptide in 99.9% D2O (1 mM) followed by incubation for 18 h at 4 °C. The selective labeling was achieved by a 50-fold dilution of fully deuterated peptides (1 mM) into cold acidic 1H2O (0.5 M acetic acid, pH 2.5, 0 °C) and immediate quenching of isotopic exchange by freezing on dry ice. Sample Preparation and MALDI Mass Spectrometry. A Bruker anchorChip MALDI sample plate with a spot diameter of 400 µm was precoated with MALDI matrix by applying 0.25 µL of 20 mg/mL DBH in 50% (v/v) acetonitrile, 0.1% (v/v) aqueous trifluoroacetic acid. After crystallization of the matrix at 25 °C, the precoated sample plate was placed in a sealed container and cooled to 4 °C. The container limits the amount of water condensation and this in turn minimizes the pump-down time of the instrument upon loading the plate. Furthermore, the low temperature of the sample plate minimizes the deuterium loss from the selectively labeled peptides to the matrix solution and crystals.6 It also improves matrix crystallization because the matrix crystals are only partially dissolved and the remaining crystals prime the crystal formation in vacuo. For MALDI ISD MS experiments, the solution containing the selectively labeled peptide was manually thawed and 1 µL was quickly applied on to the cold precoated MALDI sample plate. Subsequently, the plate was loaded into the ion source of the MALDI instrument. MALDI ISD mass spectra were acquired on a Ultraflex TOF/TOF (Bruker) equipped with a 337-nm nitrogen laser. The ISD spectra were acquired in positive reflector mode using a 25-kV acceleration voltage and 150-ns ion extraction delay unless stated otherwise. For the normal MS acquisition, the laser was operated slightly above threshold at 25 Hz and the low-mass gate was set to 300 Da. To induce ISD, the laser intensity was increased from 13 to 28% and 200 consecutive laser shots were accumulated for each spectrum. The nonlabeled controls were prepared by a 50-fold dilution of fully deuterated peptide (1 mM) into acidic 1H2O (0.5 M acetic acid, pH 2.5, 25 °C) followed by overnight incubation. The peptide is thus fully equilibrated with the solution, and the deuterium content of the peptide reflects the deuterium content of the solution (i.e., 2%). We have experimentally verified the residual deuterium content by measuring the mass increase of the intact peptides after the peptides have equilibrated with the solution. Data Analysis. All recorded spectra were manually calibrated and analyzed using FlexAnalysis v. 2.4 (Bruker). Data were

exported to Excel (Microsoft, Redmond, WA), and average masses of the obtained fragment and precursor ions were calculated as the intensity-weighted average of the masses of the isotopic envelopes. The mass increase in fragment ions obtained from labeled peptides was calculated relative to corresponding fragment ion masses of the nonlabeled peptide, which had been recorded on the same day to compensate for fluctuations in the calibration. The deuterium content of the peptide precursor was estimated from normal MS spectra recorded immediately before and after MALDI ISD. The degree of scrambling was calculated as described previously by comparing the experimentally derived deuterium levels of fragment ions to the expected theoretical values in the case of 0 and 100% scrambling.17 The residual deuterium content present during the exchange reaction was determined by the mass shift of peptide P1 after equilibration in the exchange-out solution (2%). RESULTS We have previously described two synthetic peptides P1 (HHHHHHIIKIIK) and P2 (HHHHHHIITIIT) that can be selectively labeled with deuterium at their backbone amides in the C-terminal half.16 This was achieved by having an N-terminal half (HHHHHH-) with a much higher intrinsic amide hydrogen exchange rate than that of the C-terminal half (-IIKIIK or -IITIIT). The bulky isobutyl side chains of the isoleucine residues block solvent accessibility of the neighboring amide hydrogens and thereby slow down their exchange kinetics.24 By contrast, the amide hydrogens flanked by histidine residues exhibit an accelerated rate of exchange presumably due to electrostatic effects.16 The peptides are selectively labeled by diluting a fully deuterated peptide into a cold acidic 1H2O solution. Under these conditions, the amides in the N-terminal half are completely protiated within a few seconds, while the amides in the C-terminal half retain their deuterons for a much longer period (half-life >100 min). Upon gas-phase fragmentation of these selectively labeled peptides, the deuterium content of the resulting fragment ions is a sensitive probe for the occurrence of intramolecular migration of amide hydrogens (1H/2H) (i.e., hydrogen scrambling). Here, we employ these peptides to investigate the occurrence of scrambling upon fragmentation during MALDI ISD. MALDI ISD of nonlabeled peptide P1 generated an extensive series of c- and z-fragment ions (c3-c9 and z4–z10, Figure 2). A similar fragmentation pattern was observed by ISD of peptide P2 (data not shown). Next, the selectively deuterated peptide P1 was subjected to ISD. In solution, the deuterons are exclusively located in the C-terminal half (-IKIIK) of the peptide. Thus, the deuterium level of fragment ions c3-c6 comprising the N-terminal half should be close to zero in the absence of gas-phase hydrogen scrambling. Inspection of the isotopic envelopes of c3, c5, and c6 generated by ISD of the labeled peptide clearly show that their deuterium levels are virtually identical to the nonlabeled control (Figure 2, insets). Evidently, the degree of intramolecular proton/deuteron migration during ISD fragmentation was minimal. Consistent with this result, the deuterons of the precursor ion were contained within the z7ion covering the C-terminal half of peptide (Figure 2, insets). However, although the z7-ion encompasses the C-terminal half that (24) Bai, Y.; Milne, J. S.; Mayne, L.; Englander, S. W. Proteins 1993, 17, 75– 86.

Figure 2. MALDI-TOF ISD mass spectrum of the peptide P1 (HHHHHHIIKIIK). The insets in the upper panel display isotopic distributions of four protonated fragment ions and the precursor ion (P) that were obtained from nonlabeled P1 control, while the insets in the lower panel were obtained from P1 selectively labeled with deuterium at the amides in its C-terminal half. The deuterium content of the precursor ion was 4.5 Da. Asterisked peaks indicate sodiated ions. The absence of excess deuterium in the N-terminal c-fragments (c3-c6) of the labeled peptide shows that ISD proceeds with a very low degree of amide hydrogen scrambling.

contains all the deuterons in solution,16 the deuterium content of the z7-ion was consistently somewhat lower (∼20%) than that of its precursor in replicate MALDI ISD experiments (n ) 5). Similar values were observed for the larger z-ions, i.e., z8 and z9. For example, a precursor ion containing 4.7 deuterons yielded z8 and z9 with 3.9 and 3.7 deuterons, respectively. It is important to note that this deuterium deficit is not caused by intramolecular migration of deuterons to the N-terminal half, as evidenced by the absence of any excess deuterium in the c3-c6 fragment. The most likely explanation for this deuterium deficit is the occurrence of intermolecular proton/deuteron transfer reactions between z-fragments and matrix molecules (vide infra). As shown in Figure 3, the deuterium content of the c-ions generated by ISD of the selectively labeled peptide P1 is very similar to the theoretical values for 0% scrambling. Analogous ISD experiments on selectively labeled peptide P2 yielded similar results (data not shown). Clearly, the solution labeling pattern is closely mimicked by the gas-phase data. It should be noted, however, that the deuterium level increases by more than one deuteron (∼1.3) from c8 to c9. We suggest that this reflects a local deuteron migration from the amide nitrogen of residue Lys9 to its side chain ε-amino group (note that the c8 ion contains the amide nitrogen of residue Lys9, while its side chain ε-amino group is contained within the c9 ion along with the amide nitrogen of residue Ile10). It is important to emphasize that the migration is highly localized, as evidenced by the absence of deuterons in the N-terminal fragment ions (Figure 2, insets). Note that this phenomenon was not observed upon ECD of the selectively labeled peptide P1.17 We have previously shown that collisional activation initiates amide (1H/2H) hydrogen migration in protonated peptides.11,12,16 Collisional activation is known to occur when the analyte ions are accelerated immediately after the UV laser has been fired, as they collide with neutrals in the relatively dense MALDI matrix Analytical Chemistry, Vol. 80, No. 16, August 15, 2008

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Figure 3. Deuterium content of the c-fragment ions obtained from ISD of the selectively labeled peptide P1 (filled circles, boldface line). The theoretical deuterium content in the case of 100% scrambling (open circles, thin line) and 0% scrambling (crosses, dotted line) is shown. The deuterium content of the precursor ion was 4.7. The fragmentation scheme in the lower panel displays the sequences of c- and z-fragment ions of peptide P1. The main chain amide hydrogens of residues in boldface were labeled with deuterium in solution. Note that the amide hydrogen of residue i is contained in the ci-1 fragment ion.

plume.25,26 By introducing a time delay between the laser pulse and the acceleration voltage, the MALDI plume expands and becomes less dense and hence the extent of collisional activation is reduced. ISD experiments performed at a shorter delayed extraction time (10 ns), however, yielded deuterium levels that were similar to those obtained from ISD experiments performed at the default delayed extraction time of 150 ns (data not shown). This indicates that ISD occurs on a time scale of 10 ns or less. Likewise, increasing the laser intensity from the ISD default value of 28-40% of full-scale laser intensity had no effect on deuterium levels of fragment ions produced by ISD fragmentation of selectively labeled peptide (data not shown). ISD of peptide P1 yielded several abundant protonated and sodiated y-fragments (y8-y11) (Figure 2). Similar results were observed for peptide P2 (data not shown). Unlike the c-and z-ions, the mechanistic origins of these MALDI ISD derived y-ions are more uncertain but likely involve alternate dissociation pathways of the radical precursor ion species (vide infra). In this regard, the origins of y-ions from MALDI ISD appear to be fundamentally different from those generated by fragmentation techniques based on collisional activation. This is illustrated by the different fragmentation patterns of peptide P1 that are observed in MALDI ISD and low-energy CID, respectively. The most abundant fragment ions observed in low-energy CID of peptide P1 are the b-ions, while the y-ions are of lower abundance.16,17,27 In contrast, (25) Kaufmann, R.; Kirsch, D.; Spengler, B. Int. J. Mass Spectrom. Ion Processes 1994, 131, 355–385. (26) Gabelica, V.; Schulz, E.; Karas, M. J. Mass Spectrom. 2004, 39, 579–593. (27) The b-ion series was more abundant than the y-ion series in the MALDI PSD spectrum obtained from nonlabeled peptide P1 using R-cyano-4hydroxycinnamic acid as matrix (data not shown).

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Figure 4. Deuterium content of protonated (filled circles, boldface line) and sodiated (filled triangles, boldface line) y-fragment ions obtained from ISD of the selectively labeled peptide P1. The theoretical deuterium content in the case of 100% scrambling (open circles, thin line) and 0% scrambling (crosses, dotted line) is shown. The deuterium content of the precursor ion was 4.7. The ISD fragmentation scheme in the lower panel displays the sequences of b- and y-fragment ions of peptide P1. Note that b-ions are not observed in our experiments. The main chain amide hydrogens of residues in boldface were labeled with deuterium in solution. Note that the amide hydrogen of residue i is contained in the yi fragment ion.

close inspection of the MALDI ISD spectrum (Figure 2) reveals that the b-ions are virtually absent, while the y-ions are the most abundant fragment ion type. To investigate the level of scrambling occurring during the generation of these y-ions, we measured the deuterium content of y-ions (y8-y11) from MALDI ISD of selectively labeled peptide P1 (Figure 4). Interestingly, the degree of scrambling for the sodiated y-ions (y8-y11) is very close to 0% with a nearly constant deuterium content for all four fragment ions (Figure 4). Likewise, the protonated y-ions (y8-y11) in the present study have a nearly constant deuterium content, albeit somewhat lower than those of the sodiated y-ions (Figure 4). If global scrambling was prevalent, the deuterium content would increase with fragment ion size. Thus, the observed constant deuterium content of y-ions (y8-y11) suggests very limited global hydrogen scrambling in the protonated and sodiated precursor ions. DISCUSSION A major experimental difficulty in probing the extent of intramolecular migration of amide hydrogens (i.e., hydrogen scrambling) in gaseous peptides is to prepare peptides with a selective labeling that enables an accurate assessment of the degree of hydrogen scrambling. In the present study, we have utilized peptides with a unique selective deuterium labeling to investigate the occurrence of hydrogen scrambling upon fragmentation by MALDI in-source decay. In solution, the peptides are labeled with deuterium exclusively in their C-terminal half, while their N-terminal half is protiated.16 At the onset of hydrogen scrambling, the deuterons in the C-terminal half will migrate

toward the N-terminal half and this is readily detected as a mass increase of the N-terminal fragment ions. Our results show that MALDI ISD of these selectively labeled peptides yields fragment ions that have retained their solution deuteration pattern. Thus, the deuterium content of the c3–c6 fragment ions comprising the fast-exchanging N-terminal half of peptide P1 contained a negligible amount of deuterium, thereby closely mimicking the solution labeling pattern (Figure 3). In accordance, the deuterons were localized in the complementary z-fragment ions. However, the deuterium content of the z-ions was somewhat lower than expected (∼20%), indicating a loss of deuterium during the ISD process. We attribute this deficit in deuterium to intermolecular proton/deuteron-transfer reactions between z-fragments and MALDI matrix molecules. In fact, several lines of evidence support the occurrence of ion-molecule reactions between z-ions and matrix molecules. First, the z-ions are initially formed as radical species, but hydrogen-transfer reactions from matrix molecules yield the abundant even-electron z-ions in MALDI ISD.18,23 Second, the observation of covalent complexes between DHB and z-ions provides direct evidence for the existence of intermolecular complexes between matrix and z-ions.18 This suggests that c-ions provide a more accurate measure of the deuterium incorporation pattern in solution. In addition to the c- and z-type fragment ions, MALDI ISD of the selectively labeled peptides yielded several abundant protonated and sodiated y-ions (y8-y11). The deuterium content of these y-ions shows evidence of limited hydrogen scrambling. A similar observation has been reported by Demmers et al. upon low-energy collision-induced dissociation of sodiated transmembrane-spanning peptides using an quadrupole ion trap.7 These peptides consisted of Leu-Ala sequences, that traverse the hydrophobic core of the membrane, flanked by Trp residues. Extensive global scrambling was, however, observed by Demmers et al. when lysine residues were introduced as flanking residues.7 In contrast, our results indicate that lysine residues do not induce global scrambling when sodiated peptides are fragmented by MALDI ISD. This difference is most likely due to different fragmentation mechanisms and very short activation times (ns) in MALDI ISD compared to long activation times (ms) during collision-induced dissociation in quadrupole ion traps. The low hydrogen scrambling levels observed upon MALDI ISD supports that c, z, and y-ions are generated by a very fast radical-based fragmentation mechanism. Thus, y-ion formation by the “mobile proton” mechanism is less likely to occur in MALDI ISD, as hydrogen scrambling is extensive with this mechanism.11,12,28 In conjunction, we have previously demonstrated that the selective labeling of peptide P1 is completely erased in y-ions generated by collisional activation.16 The current results indicate that the y-ions generated during MALDI ISD are formed by a hydrogen radical-transfer mechanism analogous to the y-ion formation mechanism in electron capture dissociation,18,29 as ECD occurs (28) Paizs, B.; Suhai, S. Mass Spectrom. Rev. 2005, 24, 508–548.

with limited vibrational excitation and hence a very low degree of scrambling.17 CONCLUSION We have demonstrated that MALDI ISD of selectively labeled peptides yields fragment ions that have retained their solution deuteration pattern. The low level of hydrogen scrambling for the c-ions is an intriguing finding which suggests that MALDI ISD may be utilized to measure the deuterium uptake of individual residues in peptides and small proteins. The procedure for fragmentation by MALDI ISD is extremely simple, and it can be carried out on standard MALDI-TOF instruments. The sample requirement for MALDI ISD analyses is in the picomole range, which is far below the amount required for NMR measurements of backbone amide hydrogen exchange kinetics. Furthermore, MALDI ISD analyses of intact purified proteins in the 10-20 kDa mass range have yielded relatively high sequence coverages (e.g., ∼70% for cytochrome c and 85% for apomyoglobin).30 A top-down analytical approach for hydrogen exchange studies of purified small proteins by MALDI ISD thus appears feasible. As this approach does not include a pepsin digestion step or chromatographic separation, the deuterium loss during analysis (i.e., back exhange) should be mimimal. It should be noted, however, that MALDI ISD is not a tandem mass spectrometry technique, and it is thus not possible to select a specific precursor ion for fragmentation. MALDI ISD is therefore not suited for the analysis of complex mixtures such as deuteriumlabeled peptic digests (as this would yield many fragment ions with overlapping isotopic distributions). For such digests, the most promising analytical approach with single-residue resolution appears to be separation by liquid chromatography and ionization by electrospray followed by ECD, as ECD is a tandem mass spectrometry fragmentation technique with a low level of hydrogen scrambling.17 Regardless of the above-mentioned limitation, our current findings appear promising for the use of MALDI ISD in combination with hydrogen exchange to study the exchange kinetics of purified peptides and small proteins. ACKNOWLEDGMENT We acknowledge The Danish Biotech Research Academy (DBRA), NovoZymes, The Danish Research Agency (to N.B.) and The Danish Natural Science Research Council (FNU, grant 27206-0493) for financial support. N.B. and K.D.R. contributed equally to this work.

Received for review May 1, 2008. Accepted May 22, 2008. AC800902A (29) Zubarev, R. A.; Kruger, N. A.; Fridriksson, E. K.; Lewis, M. A.; Horn, D. M.; Carpenter, B. K.; McLafferty, F. W. J. Am. Chem. Soc. 1999, 121, 2857– 2862. (30) Lennon, J. J.; Walsh, K. A. Protein Sci. 1997, 6, 2446–2453.

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