Deuterium Exchange of

Article pubs.acs.org/ac

Site-Specific Analysis of Gas-Phase Hydrogen/Deuterium Exchange of Peptides and Proteins by Electron Transfer Dissociation Kasper D. Rand,*,† Steven D. Pringle,§ Michael Morris,§ and Jeffery M. Brown§ †

The Department of Pharmaceutics and Analytical Chemistry, University of Copenhagen, Copenhagen, Denmark Waters MS Technologies Centre, Micromass U.K. Ltd., Floats Rd, Wythenshawe, Manchester M23 9LZ, U.K.

§

S Supporting Information *

ABSTRACT: To interpret the wealth of information contained in the hydrogen/deuterium exchange (HDX) behavior of peptides and proteins in the gas-phase, analytical tools are needed to resolve the HDX of individual exchanging sites. Here we show that ETD can be combined with fast gas-phase HDX in ND3 gas and used to monitor the exchange of side-chain hydrogens of individual residues in both small peptide ions and larger protein ions a few milliseconds after electrospray. By employing consecutive traveling wave ion guides in a mass spectrometer, peptide and protein ions were labeled on-the-fly (0.1−10 ms) in ND3 gas and subsequently fragmented by ETD. Fragment ions were separated using ion mobility and mass analysis enabled the determination of the gas-phase deuterium uptake of individual side-chain sites in a range of model peptides of different size and sequence as well as two proteins; cytochrome C and ubiquitin. Gas-phase HDX-ETD experiments on ubiquitin ions ionized from both denaturing and native solution conditions suggest that residue-specific HDX of side-chain hydrogens is sensitive to secondary and tertiary structural features occurring in both nearnative and unfolded gas-phase conformers present shortly after electrospray. The described approach for online gas-phase HDX and ETD paves the way for making mass spectrometry techniques based on gas-phase HDX more applicable in bioanalytical research.

E

dynamics simulations and electron capture dissociation (ECD)19 experiments have provided evidence for a stepwise unfolding of unsolvated protein ions.7,20 Furthermore, it was recently demonstrated by a similar approach that a small three-bundle helix protein can retain a native fold for several seconds after ESI if significant electrostatic stabilization occurs between native structural elements (salt bridges and ionic hydrogen bonds).21 In good agreement with such findings, larger molecular assemblies consisting of multiple individual proteins often remain intact during the transition into vacuum which has made the study of biologically relevant supramolecular protein complexes by MS an increasingly popular approach.22,23 The use of MS to measure HDX of proteins in solution has become a popular method for interrogating the conformation, dynamics, and molecular interactions of proteins to understand biological function.24−29 We and others have shown that prompt ion-electron based fragmentation methods, such as electron capture- or electron transfer dissociation30 (ECD/ETD), can be used to extract site-specific information for peptides and proteins labeled in solution.31−39 These developments enable MS analysis of biologically relevant protein states in solution at an unprecedented level of detail. In contrast, MS analysis of protein HDX in the gas-phase has so far only seen very few

lectrospray ionization (ESI)1 has revolutionized the use of mass spectrometry (MS) to characterize biomolecules in more terms than molecular mass alone. Several gas-phase techniques based on MS have been developed to directly probe the conformations of polypeptide ions. Ion mobility mass spectrometry (IMS) measures the drift time of ions traversing a background gas and relates such information to the collisional cross section of the molecule.2 Alternate MS approaches subject protein ions to gas-phase chemistry inside a mass spectrometer such as proton-transfer-,3−5 radical based-,6−8 or hydrogen/ deuterium exchange reactions9−16 and derive structural information from measured reaction kinetics. The analysis of protein ion conformers by MS has shown that unsolvated proteins can adopt a plurality of both transient solution-like conformations as well as stable non-native conformations. The relative abundance of conformers within this ensemble appears to change over time in the gas-phase. Early work by the McLafferty group used gas-phase HDX to show that cytochrome C adopts different stable conformers after equilibration in vacuum for several seconds to hours.10 At the opposite end of the time-scale, studies employing IMS have demonstrated that small globular proteins like cytochrome C or ubiquitin can retain a collisional cross-section that correlate with their solution-phase structure during several tens of milliseconds after transfer into the gasphase by ESI.17,18 A loss of native tertiary structure was observed at longer time scales (seconds) indicated by an increased population of new elongated and compact states.17,18 Molecular © 2012 American Chemical Society

Received: November 3, 2011 Accepted: January 10, 2012 Published: January 10, 2012 1931

dx.doi.org/10.1021/ac202918j | Anal. Chem. 2012, 84, 1931−1940

Analytical Chemistry

Article

HDMS mass spectrometer (Waters Corp., Milford, MA, U.S.A.). All protein and peptide samples were infused directly into the mass spectrometer via a nano-ESI needle or a fused-silica capillary using a syringe pump (Harvard Apparatus, Holliston, MA, U.S.A.) at flow rates 0.5−1.5 μL/min. The nanoflow Z-spray ESI source was equipped with a precut picotip emitter and operated with a capillary voltage of 3.5−4 kV, a sampling cone voltage of 10−20 V and a source-block temperature of 100− 130 °C. In MS-mode, the trap TWIG collision energy was set to 4 V. Collisional induced dissociation (CID) MS/MS experiments were performed by ramping the ion injection energy in the trap T-wave (Trap CE) from 4 to 28 V. Mass accuracy was achieved by calibration in MS/MS mode with 100 fmol/μL GFP. Mass spectra were acquired over an m/z range of 50−2000. ETD reagent (4-nitrotoluene or 1,3 dicyanobenzene) was introduced into a glow discharge anion source by a flow of nitrogen gas (Makeup Flow, 4.7−8.4 mL/min) across reagent crystals contained in a sealed vial. Radical anions of the ETD reagent were generated in the discharge anion source as described previously45 using a discharge current of 15−30 μA. Automated switching of ion source polarity and ion transfer optics allowed for sequential transfer of cations and anions generated in the source region to the trap TWIG for ETD. Cations were selected for ETD in the quadrupole using a mass selection window of ±3−5.0 m/z units. ETD MS/MS data was acquired over 1 s scans with an anion refill time of 100 ms between scans. Optimal ETD efficiency was achieved using a wave height of 0.2 V and a wave velocity of 250 m/s in the trap TWIG. MS scans were recorded at a trap T-wave height of 1.5 V as a higher amplitude T-wave prevents the ETD ion−ion reaction from occurring. Experiments with supplemental activation of nondissociated products of the ETD reaction was achieved by varying the ion injection energy in the transfer TWIG (“Transfer CE”) from 6 to 10, 15, or 25 V. Experiments with ion mobility separation of ETD product ions was achieved in the IMS mode of the instrument by infusing N2 gas (40 mL/min) into the mobility TWIG and optimizing the wave height (6−28 V) and wave velocity (200−400 m/s) settings for maximal ion separation. Gas-phase HDX of peptide and protein cations was performed in the source TWIG of the instrument in a similar manner as described previously13 and further detailed in the Supporting Information. Data Analysis. Mass spectra were processed with the MassLynx software (Waters Corporation, Milford, MA). Ion mobility data was processed in the Driftscope module of the MassLynx software package. The gas-phase deuterium contents of peptides, proteins, and their fragment ions were calculated in Excel (Microsoft, Redmond, WA) from intensity-weighted average masses of deuterium labeled ions relative to the corresponding masses of nonlabeled ions measured in the absence of ND3 gas. By calculating the difference in deuterium content of consecutive c- or z-fragment ions present in ETD spectra, the deuterium content of individual amino acid residues could be determined. If fragment ions were missing from a series of consecutive c- or z-type fragment ions, an average deuterium value was estimated for sites in the intervening residues by dividing the difference in deuterium content between fragment ions bordering one or more undetectable fragment ions, by the theoretical number of fast-exchanging side-chain hydrogens in the intervening amino acid residues. The experimental error of measured deuterium values was estimated from the standard

applications within biological research. We have previously described the ability to perform very rapid (subms) gas-phase HDX of peptide and protein ions within the confines of a traveling wave ion guide (TWIG)40,41 filled with ND3 gas by a simple modification to a commercially available time-of-flight mass spectrometer.13 Efficient and controlled deuterium labeling of fast-exchanging sites in different gaseous proteins was achieved a few milliseconds after ESI, thus accessing gasphase conformers that appeared to retain structural features found in solution.13,42 Gas-phase HDX using ND3 does not occur via the “relay” mechanism as do weaker bases such as D2O43 and HDX-mediated by ND3 gas may therefore more directly report on gas-phase conformation.12,13 The interpretation of the gas-phase HDX profile for even a small gaseous protein in terms of structure is, however, greatly complicated by the absence of spatially resolved labeling information. The gasphase deuterium uptake of a polypeptide ion is the aggregate of exchange rates from a multiplicity of different exchanging sites and thus contains a great wealth of information. Deciphering this information into local structural information is however a considerable challenge. Mathematical procedures have been proposed for deriving grouped rate constants from deuterium uptake curves of intact peptide ions and site-specific gas-phase HDX information was demonstrated for small peptides.12,44 However, new analytical tools are required to resolve the global gas-phase HDX of larger peptides and proteins into HDX profiles of individual exchanging sites. Here we combine gas-phase HDX and ETD mass spectrometry in an online all-MS workflow to resolve the gasphase deuterium labeling of peptides and proteins to individual amino acid residues. Peptide and protein cations are labeled a few milliseconds after ESI, in a TWIG containing ND3 gas, mass selected for ETD in a quadrupole and subjected to reaction with radical anions when traversing a neighboring TWIG. By mass analysis of produced fragment ions in the TOF-mass analyzer, incorporated deuteriums can be assigned to individual fast-exchanging sites of the labeled peptide or protein ion. Our analytical approach provides the basis for a better understanding of factors that govern site-specific HDX reactivity in gaseous peptides and proteins and should enable more detailed conformational studies of protein conformers present in the gas-phase shortly after electrospray.

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MATERIALS AND METHODS Materials. DP9 peptide (KFVGVGVGVGFK) was a generous gift from Dr. Frank Kjeldsen. All other proteins and peptides were purchased from Sigma Aldrich (St. Louis, MO) and were used without further purification. 99% deuterated ammonia gas was purchased from Cambridge Isotope Laboratories (Andover, MA) and contained in a 25 L lecture bottle fitted with a regulator (Model 322−2702-CGA180, CONCOA, Utrecht, Netherlands). Sample Preparations. Lyophilized peptides (Glu-fibrinopeptide B, angiotensin I, substance P, DP9) were dissolved in water (50−100 μM) and diluted into ESI buffer (50% acetonitrile, 0.1% formic acid) to a concentration of 1 to 10 μM. Mellitin was dissolved in ESI buffer to a concentration of 7 μM. Equine cytochrome C was dissolved in water (300 μM) and diluted to 10 μM in ESI buffer. Human ubiquitin was dissolved in water (300 μM) and diluted to 10 μM in either (a) 10 mM ammonium acetate, pH 6, or (b) ESI buffer. Mass Spectrometry. Positive electrospray ionization (ESI) mass spectrometry was performed on a Waters Synapt G2 1932



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deviation of triplicate ETD measurements (n = 3) unless stated otherwise. Relative deuterium uptake values for individual residues, plotted as percentile values in Figures 4 and 5, were determined by normalizing spatially resolved deuterium levels derived from c- and z-type fragment ions to the theoretical number of fastexchanging side-chain hydrogens (not including charging protons) in the corresponding amino acid residue or peptide segment. Likewise, error bars in Figure 4 represent the normalized standard deviation of experimentally determined deuterium values (n = 3). Error bars could not be determined for residues Thr14 and Glu16 of the 11+ ubiquitin ion in Figure 4a.

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RESULTS AND DISCUSSION Online Gas-Phase HDX and ETD of Peptide Ions. Controlled gas-phase HDX can be performed by infusing ND3 gas into any of the four TWIGs of the Q-TOF (Synapt) mass spectrometer13 (Supporting Information Figure S1). By performing the gas-phase HDX in the front-end source TWIG (Supporting Information Figure S1) of the instrument we are able to a) label peptide and protein cations at relatively high reagent gas pressures (1 × 10−4 to 1 × 10−1 mbar) immediately after ESI (1−2 ms) and b) achieve a very fast gasphase labeling step (0.1−10 ms) determined by the source Twave velocity (from 10 to 1000 m/s). The short labeling times employed allows for selective labeling of fast-exchanging hydrogens in amino acid side-chain moieties as indicated previously.13 An inherent advantage of this rapid labeling step is that gas-phase conformers that are present shortly after ESI may be probed, thus limiting any losses or rearrangements of structural elements relevant to solution.17,18,20,46 We have previously shown that gas-phase HDX in a TWIG can be combined with other gas-phase techniques such as ion mobility in neighboring TWIGs of the mass spectrometer in a transmission mode type fashion.13 Here we subjected the peptide Angiotensin I (AT1) to ND3-mediated HDX in the source TWIG and mass-selected the deuterated form of the 3+ peptide ion for ETD by reaction with 1,3 dicyanobenzene anions in the trap TWIG. ETD of 3+ AT1 ions yielded a rich series of c- and z-type fragment ions (Figure 1a) originating from the gas-phase cleavage of main-chain N−Cα bonds of the peptide (Figure 1b). No apparent reduction in ETD efficiency was observed because of the presence of labeling gas in the source TWIG. It was however noted that efficiency decreased at elevated wave height settings (>1.2 V) in the source TWIG, indicating an impaired transmission of anions through the TWIG under these settings possibly through fragmentation or electron loss. Changes in deuterium content across consecutive fragment ions of AT1 revealed site-specific labeling information (Figure 1c). Deuterium labeling only occurred at amino acid side-chain moieties with fast-exchanging hydrogens (Asp1, Arg2, Tyr4, His6, His8). Also, by comparing the deuterium content of the intact peptide to the largest fragment ions (c9 and z9), it was apparent that hydrogens at both peptide termini exchanged fully during the rapid labeling reaction. The latter was further verified by inspecting the deuterium content of the fragment ion resulting from ETD-mediated loss of ammonia from the N-terminal amino group.38 Overall, these findings are in good agreement with earlier studies9,47,48 indicating that primarily fast-exchanging sites in a peptide (i.e., heteroatom bound hydrogens at side-chains or the N- and C-termini) exchange in a millisecond time frame in ND3 gas.

Figure 1. Site-specific gas-phase HDX of angiotensin I resolved by ETD. (a) ETD mass spectrum of triply protonated AT1 peptide. The reduced species that originate from peptide ions that capture one or more electrons without dissociation are denoted by RS+ and RS2+. Gas-phase HX of the intact peptide causes a shift in mass in ETD fragment ions (c7 and z7, insert) corresponding to the local deuterium uptake into parts of the gaseous peptide. (b) Chemical structure of fragment ions resulting from backbone cleavage at the N−Cα bond of the AT1 peptide (c- and z-ions) following ETD. Potentially fast exchanging hydrogens (labile on side-chain moieties and the C- and N-termini) are labeled (red). (c) Deuterium contents measured in cand z-fragment ions generated by ETD. The standard deviation of three replicate experiments is indicated by error bars. Changes in deuterium content across consecutive fragment ions reveals a selective labeling of fast exchanging sites of the peptide. 1933



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spanning the strongly apolar environment of a lipid membrane.50 Thr residues in a gas-phase helix are likely to have a similar propensity for forming an intrahelical hydrogen bond between the hydroxyl group of the side-chain and a main-chain amide group, which could protect this hydrogen from gas-phase HDX. Differences in gas-phase HDX were also observed for Lys residues. The synthetic model peptide DP9 (KFVGVGVGVGFK) incorporated 3 deuteriums at each Lys side-chain moiety, which identifies these two side-chains as charge carriers in this 2+ peptide ion. Interestingly, the intact singly protonated form of the peptide (1+ DP9) incorporated only 0.5 deuterium under the same conditions (data not shown), strongly indicating the presence of structural features in the 1+ DP9 ion which in turn are lost due to Coulombic repulsion in the 2+ DP9 ion. The Lys residues in the 3+ Substance P ion (Lys3) and the 4+ Mellitin ion (Lys21 and Lys23) exchanged readily, incorporating approximately 2 deuteriums each. In stark contrast, Lys7 of the 4+ Mellitin ion exchanged only 0.5 D. Experimental evidence has suggested the formation of interactions between Lys7 and backbone carbonyl oxygens51 or even polar groups at the C-terminal end (Gln25 and Gln26) in the 4+ Mellitin ion.49 The reduced gas-phase HDX of Lys7 relative to Lys22 and Lys24 could correlate with an engagement of Lys7 in electrostatic interactions. The absence of similar interactions for Lys22 and Lys24 could in turn be due to the absence of charges on these sites making solvation to backbone carbonyl oxygens unlikely. Thus, the site-specific HDX of Lys7, Thr10, and Thr11 could report on a specific conformation adopted by the 4+ Mellitin ion in the gas-phase. Arginine residues also exchanged to varying extents in the different peptides studied. Arg22 and Arg24 of the 4+ Mellitin ion exchanged the most, incorporated 3 deuterium each (both charge carriers), suggesting that these side-chains protrude from the C-terminal helix of this peptide and do not engage in solvation with backbone carbonyl oxygens. In good agreement, electrostatic repulsion due to the close proximity of the two charges on Arg22 and Arg24,51 make internal backbone solvation and hydrogen bonding unlikely. The single Arg residues in AT1 and Substance P exchanged only 1 and 1.8 respectively. In conjunction, HDX studies of other small peptides containing Arg residues using ND3 gas has indicated the existence of charge-solvated gas-phase structures that shield hydrogens on the Arg guanidinium group from gas-phase HDX.52−54 A more detailed investigation of the correlation between the gas-phase HDX of these peptides and their gas-phase structure

Deuterium uptake at individual sites in AT1 could be tracked as a function of labeling time or ND3 gas pressure in the source T-wave (Supporting Information Figure S2). Interestingly, some side-chain moieties containing inherently fast-exchanging hydrogens did not exchange even at high pressure and maximal labeling times (10 ms). For instance, Arg2 incorporated only 1 deuterium out of 4 possible hydrogens (excluding the charge carrier). Likewise, His6 and His8 incorporated 0.6 and 2 deuteriums, respectively, corresponding to full HDX of labile hydrogens of the imidazole side-chain of His8, including a charging proton, but incomplete HDX of the hydrogen on the imidazole side-chain of His6. It is tempting to assign such protection from HX of particular side-chain hydrogens to ion structure, but considering the size and the charge state of the AT1 peptide ion (+3), these differences may also reflect other factors such as gas-phase basicity.43,47 Gas-phase HDX and ETD experiments on a range of other peptides of differing size and mass-to-charge ratio (Substance P, DP9, Glu-Fibrinopeptide B, Mellitin) is summarized in Table 1. Overall, peptide ions subjected to HDX at the same conditions for maximal labeling (10 ms in 1 × 10−2 mBar ND3 gas) selectively incorporated deuteriums into the side-chain moieties of Asp, Glu, Ser, Thr, Tyr, His, Lys and Arg in addition to the N- and C-termini. The significantly slower exchanging amide groups of the peptide backbone or the side-chains of Asn or Gln were not labeled. As observed with AT1, the degree to which exchangeable side-chain hydrogens exchanged in a given peptide was often quite different. For instance, the carboxyl hydrogen of Asp1 in the 3+ AT1 ion exchanged to unity, whereas the two Asp and Glu residues of the 2+ GluFibrinopeptide B ion exchanged only partially (0.6). Notably, other experimental evidence has indicated that these two acidic residues in Glu-Fibrinopeptide B engage in hydrogen bonds with the C-terminal arginine (Arg14) to define the conformation of this doubly protonated peptide ion in the gas-phase.49 The Ser residue of the 4+ Mellitin ion (Ser18) exchanged to unity while the two Thr residues (Thr10 and Thr11) exchanged only partially. 4+ Mellitin ions have been proposed to retain structural features from solution characterized by an N-terminal and a C-terminal helix with Pro13 functioning as a hinge between these two structures.49 The reduced gas-phase HDX of both Thr10 and Thr11 could correlate with the engagement of these residues in a stable helical segment in the gas-phase. Interhelical hydrogen bonding has been observed in nearly 100% of Thr residues in transmembrane helices

Table 1. Residue-Specific Deuterium Contents of Peptide Ions Following Fast (10 ms) Gas-Phase HDX in ND3 Gas polypeptide name no. of residues m/z intact peptide Asp Glu Ser Thr Tyr His Lys Arg Trp N-terminal −NH2 C-terminal −COOH

angiotensin I 10 649.3 (3+) 8.6 1 (D1)

substance P 11 450.6 (3+) 6.8

KFVGVGVGVGFK 12 597.7 (2+) 8.5

Glu-fibrinopeptide B 14 786.3 (2+) 9 0.6 (D5) 0.6 (E1)/? (E7, E8) ? (S12)

mellitin 26 712.9 (4+) 16.4

1 (S18) 0.5 (T10, T11)

1 (Y4) 0.6 (H6) / 2 (H9) 2 (K3) 1.8 (R1)

3 (K1) / 3 (K12)

1 (R2) 2 1

2 1

2 1

? (R14)

1934

2 ?

0.5 (K7) / ≥2 (K21, K23) 3 (R22)/3 (R24) ? (W19) 1 1



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can similarly be applied to increase the abundance of ETD fragment ions of larger peptides or proteins. Gas-Phase HDX and ETD of Protein Ions. We next investigated the ability of the combined gas-phase HDX and ETD approach to extract site-specific HDX information for protein ions. ETD of intact 11+ ubiquitin ions, produced by ESI at denaturing solution conditions, produced a rich but complex mass spectrum containing an extensive series of c- (c1−c40) and z-type (z3−z48) fragment ions of the 76-residue protein. Some fragment ions failed to produce suitable ion statistics, while spectral overlap between fragment ions of similar m/z value impaired the mass analysis of several others. We therefore investigated the use of ion mobility separation of gas-phase labeled ETD fragment ions prior to mass analysis. During this three-step MS experiment, ubiquitin ions generated in the ESI source were labeled in the source TWIG, massselected in the quadrupole, fragmented by ETD in the trap TWIG and resulting product ions propelled through a neighboring gas-filled (mobility) TWIG before entering the TOF mass analyzer (Supporting Information Figure S1). Separation of deuterium labeled ETD fragment ions of ubiquitin was achieved by tuning the amplitude and speed of the T-wave propelling the fragment ions through the mobility TWIG containing N2 gas (0.1 mBar). Ion mobility readily resolved ions of differently charged c- and z-type fragment ions of deuterium labeled ubiquitin, thus greatly reducing signal overlap in resulting mobility-resolved mass spectra (Figure 2). Such a combination of ETD and IMS could also be of use in other “topdown”-type applications.32,35,55,56 Experimentally determined deuterium levels of c- and z-type fragment ions from ETD of 11+ ubiquitin ions following gasphase HDX at conditions for optimal labeling efficiency are shown in Figure 3. The deuterium content increased with fragment ion size in a nonlinear fashion corresponding to sitespecific incorporation of deuterium. As observed in experiments using smaller peptides, the deuterium content was constant across consecutive fragment ions if these did not contain additional amino acid residues with fast-exchanging side-chain hydrogens. For instance, the deuterium level for fragment ions z14−z17 was constant at 7.3 D showing that residues Asn60Ile61-Gln62 of the 11+ ubiquitin ion did not incorporate any deuterium during the gas-phase labeling step whereas the deuterium level increased to 8.3 for z18 showing that the sidechain hydroxyl group of Tyr59 exchanged completely, taking up 1 deuterium (Figure 3). Mass analysis of the 11+ ubiquitin ion showed that the intact protein incorporated a total of 43 deuteriums (out of 67 expected fast-exchanging hydrogens based on the protein sequence) at the employed conditions for maximal labeling (10 ms in 1 × 10−2 mBar ND3 gas). The summed deuterium content of fragment ions c41 (residues 1−41) and z35 (residues 42−76) was 42.9 D indicating a good reproducibility for the gas-phase HDX-ETD analysis. Gas-phase HDX and ETD experiments were also performed on cytochrome C, a larger 104-residue protein. ETD of intact 16+ cytochrome C ions, labeled on-the-fly by ND3 gas in the source TWIG, produced a series of z-type fragment ions (z5−z52) covering the C-terminal half of the protein. As with ubiquitin, a segmental increase in the deuterium level of z-type ions was observed with fragment ion size indicating selective labeling of fast-exchanging side-chain sites in the gaseous protein (Supporting Information Figure S4). ETD of cytochrome C failed to yield more than a few c-type fragment ions (c4−c9, Supporting Information Figure S4) thus limiting

is beyond the scope of the present report. However, the experiments on peptides of different size and sequence demonstrate the general feasibility of fast online gas-phase HDX and ETD experiments to dissect the exchange behavior of side-chain sites in gaseous peptides and suggests that such information could be sensitive to structural features in gaseous peptide ions. Insights into Peptide Fragmentation from Site-Specific Gas-Phase HDX Analyses. ETD of AT1 also generated several a- (a2, a3, a4) and y-type (y2, y3, y7) fragment ions. These product ions are the result from an alternate reaction pathway involving cleavage of the -Cα-CO- bond as well as the −CO-NHpeptide bond (Supporting Information Scheme S1). The a- and y-type ions replicated the site-specific deuterium labeling pattern found in corresponding c- and z-type ions (data not shown) which confirms their origins with the ETD reaction. We observed that the deuterium content of a-type fragment ions of AT1 was consistently 1 deuterium lower than the corresponding c-type fragment ions (comparison of a2 to c2, a3 to c3 etc). Likewise, the deuterium contents of y-type fragment ions was 1 deuterium higher than the corresponding z-type fragment ion (comparison y2 to z2, y3 to z3 etc). This finding supports the proposed mechanism for ETD30 where a hydrogen (or a deuterium) from a nearby basic site (Arg or Lys), situated C-terminal to the cleavage site, is relocated to the resulting c-type fragment ion after dissociation. In contrast, the donated hydrogen from the N-terminal basic site is retained in y-type fragment ions in accordance with the proposed mechanism (Supporting Information Scheme S1). Similar observations were made during later gas-phase HDX-ETD experiments on protein ions (see later in the text and Supporting Information). We note that the ability to deuterate side-chain sites in gaseous polypeptides and subsequently track the relocation of deuterium from side-chain moieties by ETD could be of general use to provide insights into fragmentation mechanisms or other type of gas-phase reactions involving peptide or protein ions (see also Supporting Information). A key technical requirement for the use of ETD to obtain site-specific deuterium information is that collisional activation of ions is minimized to prevent gas-phase perturbation (i.e., hydrogen scrambling) of the deuterium label prior to dissociation.31 The presence of deuterium exclusively at fast-exchanging side-chain sites in all peptides studied demonstrate the absence of scrambling of the deuterium label during the present gasphase HDX-ETD experiments. If such scrambling occurred, deuterium should be observed at residues without fastexchanging side-chain hydrogens (for instance, Gly, Ala, Val, Ile, Phe etc.) because of the gas-phase migration of deuterium from side-chain positions to main-chain amide positions in the activated peptide ion. We verified this by gas-phase HDX-ETD experiments where 2+ Glu-Fibrinopeptide B ions, labeled with deuterium in the gas-phase, were subjected to either ETD or CID in the trap TWIG (Supporting Information Figure S3). Extensive randomization of the gas-phase deuterium label was observed in CID fragment ions of Glu-Fibrinopeptide B in contrast to corresponding ETD fragment ions (Supporting Information Figure S3). Furthermore, we found that mild supplemental activation in the transfer TWIG af ter ETD to increase fragment ions yields of either gas-phase labeled Glu-Fibrinopeptide B or AT1, did not perturb the site-selective deuteration pattern in resulting ETD fragment ions (data not shown). This is in good agreement with earlier work on peptides labeled with deuterium in solution.39 The present findings suggest that a strategy of mild supplemental activation during gas-phase HDX-ETD experiments 1935



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Figure 2. Enhanced detection of site-specific gas-phase HDX by ion mobility separation of ETD generated fragment ions. ETD spectra of fragment ions of unlabeled (a) and gas-phase deuterium labeled (b−d) 11+ ubiquitin ions without (a, b) or with (c, d) ion mobility separation of ETD fragment ions. Online transmission mode gas-phase HDX/ETD/IMS experiments were performed in three sequential TWIGs of the mass spectrometer.

experimental evidence indicate the formation of α-helical structures in gas-phase equilibrated ubiquitin ions.5,7,62 The inherent ability of the α-helix for internal solvation of polar and charged sites during solvent removal appears to make α-helices prevalent in equilibrated protein ions of low to medium charge density.63 HDX protected sites in the 11+ ubiqutin ion colocalize to four regions of the protein (Figure 4). Reduced HDX of the side-chain hydroxyl groups of Thr9, Thr12, Thr14, Thr55, and Ser57 could be due to the presence of α-helical structures in these regions that allow favorable intrahelical hydrogen bonding of side-chain hydroxyl groups (as also indicated earlier for Thr residues of the 4+ mellitin ion). Notably, gas-phase measurements on 11+ ubiquitin ions using ECD and photofragment spectroscopy62 support the presence of helices in regions that encompas the Thr and Ser residues of ubiquitin presently protected from gas-phase HDX. In contrast, Thr7, Thr22, and Thr66 were not similarly protected and exchanged readily, which suggests that these residues are not situated in α-helical structures of the 11+ protein ion but rather in loops or other nonhelical elements. The low relative deuterium uptake of Lys11, Asp39, Arg42, Arg54, Arg72, and Arg74 could in turn be due to interhelical tertiary contacts (electrostatic interactions) that have been proposed to occur in several thermalized multiply protonated states of ubiquitin.62 For instance, data from ECD of 7+ and 8+ ubiquitin ions equilibrated in the gas-phase for 20 s, supported the presence of one or more electrostatic interactions involving Lys11, Asp39, Arg42, Arg54, Arg72, and Arg74.62 Long-range interactions in unsolvated polypeptide ions are mainly due to electrostatic interactions, as these are enhanced in vacuum relative to solution, and thus can dictate gaseous polypeptide structures.63 It was proposed that tertiary contacts were destabilized in higher charges states of ubiquitin (including the 11+ ion)62 but the presence of such contacts is likely to depend on the experimental conditions and the time scale of the measurement and thus tertiary contacts could occur in 11+ ubiquitin ions in the millisecond time frame right after ESI accessed by our experimental approach. Alternatively, the observed protection of fast-exchanging sites on some Lys and Arg residues of 11+ ubiquitin while not on others, could be due to local self-solvation of charged sites with mainchain carbonyl oxygens in gas-phase helices, a common feature in equilibrated gaseous protein ions.21,62

site-specific HDX analysis of the N-terminal half of the protein. The absence of c-type fragment ions was likely due to the presence of the heme group that coordinate covalently to Cys14 and Cys17 in the N-terminal end of the protein.57 Protein size and the presence of disulfide bonds or other covalent modifications can limit top-down ECD/ETD applications58 and these factors therefore also limit the ability of the current gas-phase HDX-ETD approach to obtain local HX information for some proteins. Nonetheless, the ETD data on gas-phase labeled cytochrome C showed that most sidechain hydrogens of the C-terminal half exchanged fully. This could correlate with the expected absence of tertiary structure in highly protonated gaseous cytochrome C ions59 formed during ESI at the employed denaturing solution conditions (see Supporting Information for a further discussion). ETD measurements on gas-phase labeled 11+ ubiquitin ions showed that protection of side-chain sites from gas-phase HDX was significantly more widespread for this protein ion. To compare site-specific HDX patterns across ubiquitin, measured site-specific deuterium levels where normalized to the theoretical number of fast-exchanging sites in each particular amino acid, not including charging protons (see also Experimental Section and Supporting Information). This relative deuterium uptake facilitated direct comparison between the HDX of different side-chain sites. Several Lys, Arg, Thr residues but also two Ser and Asp sites of the 11+ ubiquitin ion showed relative deuterium uptake levels significantly below 50% during gasphase HDX at conditions for either partial or maximal deuterium labeling (Figure 4a and 4b). Earlier studies of the HDX of cytochrome C and ubiquitin in solution have shown that ubiquitin folds in a highly cooperative and rapid manner (10−20 ms),60 while cytochrome C folds through several slower-folding events (100 ms-10s).61 The increased protection from gas-phase HDX of side-chain sites in ubiquitin relative to cytochrome C could indicate that while both proteins were ionized from the same denaturing solution conditions, ubiquitin adopts hydrogen-bonded structures more readily than cytochrome C on a millisecond time frame in the gas-phase, in apparent correlation with the ability of these two proteins to fold under native conditions in solution. Ubiquitin is a highly stable β-sheet protein under native solution conditions, but convincing 1936



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Figure 4. Site-specific gas-phase deuterium uptake of ubiquitin ions as measured by ETD. The relative deuterium uptake for a given residue was determined from the difference in deuterium contents of adjacent c- or z-type ETD fragment ions of gas-phase labeled 11+ ubiquitin ions normalized to the number of labile side-chain hydrogens in that residue (not including charging protons). Data was recorded at (a) partial (30 D in intact protein) and (b) maximal (43 D in intact protein) labeling conditions in the source TWIG. The standard deviation from three replicate experiments is indicated by error bars. Shaded regions of the charts indicate regions where residues displayed protection from exchange with a relative deuteration level ≤50%.

showed that most fast-exchanging side-chain sites exchanged readily (>75%) (Figure 5). Interestingly, several of the Thr residues (Thr9, Thr12, Thr14) that were protected from HDX in the 11+ ion exchanged fully in the 6+ ion under similar conditions for maximal exchange in the source TWIG. Inspection of the structure of native ubiquitin from solution66 showed that these Thr residues form part of an outer strand of a β-sheet which thus expose Thr side-chain hydroxyl groups and do not allow them to engage in internal hydrogen bonding. The Thr residues would thus be expected to exchange readily if this solution structure was retained in the 6+ ion during the fast gas-phase HDX step a few milliseconds after ESI. Similarly, residues Arg72 and Arg74, which were strongly protected in the 11+ ion (0−25%), exchanged readily in the 6+ ubiquitin ion (>75%). Arg72 and Arg74 are situated in the highly surface exposed and unstructured C-terminal in the native solution structure. Their gas-phase reactivity in the 6+ ion thus appears to correlate with an ubiquitin ion that retains an overall native fold. Only two sites (Tyr59 and Ser65) displayed reduced gasphase HDX in the 6+ ubiquitin ion (75%) in the 11+ charge state of ubiquitin under similar exchange conditions. Other gas-phase techniques have shown that even small globular proteins can retain condensed-phase structures in the gas-phase after ESI but unfolds to more elongated conformers after 30−60 ms.17,18,20 While the absence of fragment ion data for the entire ubiquitin 6+ ion prevent a more extensive comparison of gas-phase HDX behavior with the known structure from solution, we note that the HDX of side-chain hydrogens in the 6+ ubiquitin ion, a few milliseconds after ESI, appear to correlate with the native solution structure of ubiquitin.

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CONCLUSIONS Here, we show the feasibility of combining gas-phase HDX with ETD in an integrated and online MS workflow. By a rapid gasphase reaction with ND3 gas, we selectively labeled fastexchanging side-chain hydrogens in different peptides and proteins. ETD in an adjacent TWIG of the mass spectrometer enabled the global deuterium uptake of both peptides and proteins to be spatially resolved to individual side-chain sites through mass analysis of fragment ions. Determination of deuterium levels in fragment ions of intact proteins labeled in the gas-phase was improved by ion mobility separation of ETD products in a third TWIG or supplemental activation in a fourth TWIG prior to mass analysis. A limitation of the current gas-phase HDX-ETD setup is that the exchange of amide hydrogens of the peptide backbone cannot be probed as ions are labeled on-the-fly during a few milliseconds. It is however envisaged that firmware modifications to the T-wave functions of the current setup could be employed to increase storage time and hence labeling time. Other setups do allow prolonged storage of polypeptides extending from seconds to several hours.10,11 On the other hand, an inherent advantage of a fast labeling step is that gas-phase protein conformers present shortly after ESI may be probed thus limiting gas-phase rearrangements of solution-phase secondary- or tertiary structure. Site-specific HDX of several of the peptide and

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ASSOCIATED CONTENT

S Supporting Information *

Instrumental setup for rapid gas-phase hydrogen/deuterium exchange, (2) mechanistic details revealed by ETD of deuterium labeled AT1, (3) site-specific HDX data on cytochrome C, and (4) the use of ETD fragment ions to determine deuterium levels in proteins. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

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ACKNOWLEDGMENTS We thank Frank Kjeldsen for providing the model peptide DP9 and useful discussions, John R. Engen for comments, and Peter Milland and Geoff Gerhardt for support. K.D.R gratefully acknowledges financial support from the Human Frontiers Science Programme, the Marie Curie Actions programme of the E.U (grant no. PCIG09-GA-2011-294214) and the Danish 1938



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Council for Independent Research | Natural Sciences (Steno grant, no. 11-104058).

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NOTE ADDED IN PROOF Very recently, Pan et al.67 has shown the use of gas-phase HDX at longer timescales (0.5−6 s) in a hexapole collision cell followed by electron capture dissociation to investigate the conformation of two gaseous peptides.

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Deuterium Exchange of

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