In ESI‐source H/D exchange under atmospheric pressure for peptides and proteins of different molecular weights from 1 to 66 kDa: the role of the temperature of the desolvating capillary on H/D exchange

Transition of proteins from the solution to the gas phase during electrospray ionization remains a challenging problem despite the large amount of attention it has received during the past few decades. One of the major questions relates to the extent to which proteins in the gas phase retain their condensed phase structures. We have used in‐electrospray source hydrogen/deuterium exchange to determine the number of deuterium incorporations as a function of protein mass, charge state and temperature of the desolvating capillary where the reaction occurs. All experiments were performed on a Thermo LTQ FT Ultra equipped with a 7‐T superconducting magnet. Ions were generated by an IonMax Electrospray ion source operated in the positive ESI mode. Deuterium exchange was performed by introducing a droplet of D₂O beneath the ESI capillary. We systematically investigated gas phase hydrogen/deuterium (H/D) exchange under atmospheric pressure for peptides and proteins of different molecular weights from 1 to 66 kDa. We observed that almost all proteins demonstrate similar exchange rates for all charge states and that these rates increase exponentially with the temperature of the desolvating capillary. We did not observe any clear correlation of the number of H/D exchanges with the value of the cross section for a corresponding charge state. We have demonstrated the possibility of performing in‐ESI source H/D exchange of large proteins under atmospheric pressure. The simplicity of the experimental setup makes it a useful experimental technique that can be applied for the investigation of gas phase conformations of proteins. Copyright © 2015 John Wiley & Sons, Ltd.     

Conformational changes of ubiquitin during electrospray ionization as determined by in‐ESI source H/D exchange combined with high‐resolution MS and ECD fragmentation

In the paper, we have demonstrated the possibility of performing hydrogen/deuterium (H/D) exchange of proteins in the region of gas‐phase ion formation in an electrospray ion source by saturating the electrospray ionization source with vapors of a deuterating agent (D₂O or MeOD). In this region, charged droplets are shrinking and the protein ions transfer into the gas phase. As a model protein, we have used ubiquitin whose ion mobility spectrometry and gas‐phase H/D exchange in the vacuum part of a mass spectrometer demonstrated the presence of gas‐phase conformers with different cross sections and H/D exchange rates. In our experiments, we observed monomodal deuterium distributions for all solvents, charge states, desolvating capillary temperature and types of deuterating agent. Also, we found that the number of H/D exchanges increases with an increasing desolvating capillary temperature and decreasing charge state. We observed that solution composition (49 : 50 : 1 H₂O : MeOH : formic acid or 99 : 1 H₂O : formic acid) influences the charge‐state distribution but did not change the degree of H/D exchange for the same charge state. Electron‐capture dissociation fragmentation shows that higher charge states contain a segment that is protected from access by the deuterating agent. Copyright © 2014 John Wiley & Sons, Ltd.     

Effects of electrospray capillary temperature on amide hydrogen exchange

Amide hydrogen/deuterium (H/D) exchange coupled with proteolysis, high-perfeomance liquid chromatographic (HPLC) separation and mass spectrometry (MS) has become a powerful tool to study protein dynamics in solution. Prior to the execution of H/D exchange experiments, various experimental parameters have to be set, including proteolysis, HPLC, and MS conditions. Here we investigate the effects of electrospray capillary temperature on deuterium retention in backbone amides of various pepsin-generated cytochrome c peptides. Lower capillary temperature generally helps retain more deuterium than higher capillary temperature. When the capillary temperature was 150 degrees C, on average 26% more deuterium was retained than when the capillary temperature was set at 250 degrees C. The effects of capillary temperature varied depending on the ions monitored. There was little difference in deuterium retention among different charge state species of the same peptide at 150 degrees C. However, a lower charge state ion loses more deuterium atoms going from 150 degrees C to 250 degrees C than the corresponding higher charge state species. These results indicate that the capillary temperature should be optimized not only to maximize the signal-to-noise of each ion followed in H/D exchange experiments, but also to minimize the deuterium loss of the ions. Also the loss of deuterium in several ions, especially lower charge state ones, should be monitored in the optimization, as the temperature effects vary among ions and are more significant for lower charge state ions.     

Improving hydrogen/deuterium exchange mass spectrometry by reduction of the back-exchange effect

The measurement of deuterium incorporation kinetics using hydrogen/deuterium (H/D) exchange experiments is a valuable tool for the investigation of the conformational dynamics of biomolecules in solution. Experiments consist of two parts when using H/D exchange mass spectrometry to analyse the deuterium incorporation. After deuterium incorporation at high D(2)O concentration, it is necessary to decrease the D(2)O concentration before the mass analysis to avoid deuterium incorporation under artificial conditions of mass spectrometric preparation and measurement. A low D(2)O concentration, however, leads to back-exchange of incorporated deuterons during mass analysis. This back-exchange is one of the major problems in H/D exchange mass spectrometry and must be reduced as much as possible. In the past, techniques using electrospray ionization (ESI) had the lowest back-exchange values possible in H/D exchange mass spectrometry. Methods for the measurement of H/D exchange by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) that have been developed since 1998 have some significant advantages, but they could not achieve the back-exchange minima of ESI methods. Here, we present a protocol for H/D exchange MALDI-MS which allows for greater minimization of back-exchange compared with H/D exchange ESI-MS under similar conditions.     

The methanol-induced conformational transitions of β-lactoglobulin, cytochrome c, and ubiquitin at low pH: A study by electrospray ionization mass spectrometry

The methanol-induced conformational transitions under acidic conditions for beta-lactoglobulin, cytochrome c, and ubiquitin, representing three different classes of proteins with beta-sheets, alpha-helices, and both alpha-helices and beta-sheets, respectively, are studied under equilibrium conditions by electrospray ionization mass spectrometry (ESI-MS). The folding states of proteins in solution are monitored by the charge state distributions that they produce during ESI and by hydrogen/deuterium (H/D) exchange followed by ESI-MS. The changes in charge state distributions are correlated with earlier studies by optical and other methods which have shown that, in methanol, these proteins form partially unfolded intermediates with induced alpha-helix structure. Intermediate states formed at about 35% methanol concentration are found to give bimodal charge state distributions. The same rate of H/D exchange is shown by the two contributions to the bimodal distributions. This suggests the intermediates are highly flexible and may consist of a mixture of two or more rapidly interconverting conformers. H/D exchange of proteins followed by ESI-MS shows that helical denatured states, populated at around 50% methanol concentration, transform into more protected structures with further increases in methanol concentration, consistent with previous circular dicroism studies. These more protected structures still produce high charge states in ESI, similar to those of the fully denatured proteins.     

Gas phase hydrogen/deuterium exchange of arginine and arginine dipeptides complexed with alkali metals

The hydrogen/deuterium (H/D) exchange of protonated and alkali-metal cationized Arg-Gly and Gly-Arg peptides with D(2)O in the gas phase was studied using electrospray ionization quadropole ion trap mass spectrometry. The Arg-Gly and Gly-Arg alkali metal complexes exchange significantly more hydrogens than protonated Arg-Gly and Gly-Arg. We propose a mechanism where the peptide shifts between a zwitterionic salt bridge and nonzwitterionic charge solvated conformations. The increased rate of H/D exchange of the alkali metal complexes is attributed to the peptide metal complexes' small energy difference between the salt-bridge conformation and the nonzwitterionic charge-solvated conformation. Implications for the applicability of this mechanism to other zwitterionic systems are discussed.     

In electrospray ionization source hydrogen/deuterium exchange LC-MS and LC-MS/MS for characterization of metabolites

A new method is described for performing hydrogen/deuterium (H/D) exchange in an electrospray ionization (ESI) source. The use of liquid chromatography (LC)-mass spectrometer equipped with an ESI source and deuterium oxide (D2O) as the sheath liquid allows H/D exchange experiments to be performed on-line. This directly provides information for determining the number and position of exchangeable hydrogens, aiding in the elucidation of the structures of drug metabolites. To demonstrate the utility of this method, LC-mass spectrometry (MS) and LC-MS/MS experiments were performed using either H2O or D2O as sheath liquid on a matrix metalloprotease (MMP) inhibitor (PD 0200126) and its metabolites. Examination of the mass shift of the deuteriated molecule from that of the protonated molecule allowed the number of exchangeable protons to be determined. Interpretation of the production-spectra helped to determine the location of the exchanged protons and assisted in the assignment of the site(s) of modification for each metabolite.     

A fast flow tube study of gas phase H/D exchange of multiply protonated ubiquitin

An electrospray ionization (ESI)/fast-flow technique has been applied to the study of gas phase hydrogen/deuterium (H/D) exchange kinetics. Multiply charged ubiquitin ions [ubiquitin + nH](n)(+), in charge states n = 7-13, were reacted with ND(3). The behavior of ND(3) as exchange reagent is different from that of the previously studied reagents, D(2)O and CH(3)OD. Contrary to those, the maximum number of exchanged hydrogen atoms and the overall exchange rate were observed to increase with increasing charge state of the ubiquitin ions. The results are reagent-dependent because the exchange mechanisms are different for the different reagents. This observation is in agreement with a recent conclusion by Beauchamp and co-workers that contrary to the assumption often expressed in earlier studies, H/D exchange kinetics may not directly reflect ion structures. The results for all three reagents are, however, consistent with observations of previous ion mobility experiments that with increasing charge state the conformers change from more compact, partially folded structures to elongated nearly linear ones. H/D exchange of (ubiquitin + 13H)(13+) with ND(3) leads to two separated ion populations reflecting the possible existence of two conformers with different exchange rates. The ions (ubiquitin + 8H)(8+) and (ubiquitin + 11H)(11+) represent a partially folded structure and an unfolded structure, respectively, and were studied in greater detail. The relative abundances of ions were measured in steps of 0.5 m/z (mass-to-charge ratio), as a function of the ND(3) flow rate. The experimental results were simulated by computer fitted curves based on a recently developed algorithm. The algorithm allows the extraction of sets of grouped rate constants. Eight rate constant groups were deduced for each of the two ions. These rate constants correspond to 32 and 44 H/D exchanges for the 8+ and 11+ charged ions, respectively. The results indicate higher individual rates for most of the exchanged atoms in the 11+ ion compared to the 8+ ion.     

Evidence for site‐specific intra‐ionic hydrogen/deuterium exchange in the low‐energy collision‐induced dissociation product ion spectra of protonated small molecules generated by electrospray ionisation

The experimental investigation of site‐specific intra‐ionic hydrogen/deuterium (H/D) exchange in the low‐energy collision‐induced dissociation (CID) product ion spectra of protonated small molecules generated by electrospray ionisation (ESI) is presented. The observation of intra‐ionic H/D exchange in such ions under low‐energy CID conditions has hitherto been rarely reported. The data suggest that the intra‐ionic H/D exchange takes place in a site‐specific manner between the ionising deuteron, localised at either a tertiary amine or a tertiary amine‐N‐oxide, and a γ‐hydrogen relative to the nitrogen atom. Nuclear magnetic resonance (NMR) spectroscopy measurements showed that no H/D exchange takes place in solution, indicating that the reaction occurs in the gas phase. The compounds analysed in this study suggested that electron‐withdrawing groups bonded to the carbon atom bearing the γ‐hydrogen can preclude exchange. The effect of the electron‐withdrawing group appears dependent upon its electronegativity, with lower χ value groups still allowing exchange to take place. However, the limited dataset available in this study prevented robust conclusions being drawn regarding the effect of the electron‐withdrawing group. The observation of site‐specific intra‐ionic H/D exchange has application in the area of structural elucidation, where it could be used to introduce an isotopic label into the carbon skeleton of a molecule containing specific structural features. This could increase the throughput, and minimise the cost, of such studies due to the obviation of the need to produce a deuterium‐labelled analogue by synthetic means. Copyright © 2010 John Wiley & Sons, Ltd.     

Liquid chromatography/quadrupole time‐of‐flight mass spectrometry in combination with online hydrogen/deuterium exchange technique for structural elucidation of phase I metabolites of iso‐phenylcyclopentylamine in rat bile

MS/MS experiment and accurate mass measurement are powerful tools in metabolite identification. However, sometimes these data do not provide enough information to assign an unambiguous structure to a metabolite. In combination with MS techniques, hydrogen/deuterium (H/D) exchange can provide additional information for structural elucidation by determination of the number of exchangeable hydrogen atoms in a structure. In this study, the principal phase I metabolites of iso‐phenylcyclopentylamine in rat bile were identified by high‐performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐Q‐TOF‐MS). Since N‐oxidation may occur because of the existence of the primary amino group in the structure, it was difficult to differentiate the hydroxylated metabolites from N‐oxides by ESI‐Q‐TOF‐MS alone. Therefore, online H/D exchange technique was applied to solve this problem. Finally, 25 phase I metabolites were detected and structurally described, in which 11 were confirmed to be N‐oxides. This study demonstrated the effectiveness of high‐resolution mass spectrometry in combination with an online H/D exchange technique in rapid identification of drug metabolites, especially in discriminating hydroxylated metabolites from N‐oxides. Copyright © 2014 John Wiley & Sons, Ltd.     

Dimerization of ionized 4‐(methyl mercapto)‐phenol during ESI,APCI and APPI mass spectrometry

A novel ion/molecule reaction was observed to occur under electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo ionization (APPI) conditions, leading to dimerization of ionized 4‐(methyl mercapto)‐phenol followed by fast H^· loss. The reaction is particularly favored during ESI, which suggests that this ion/molecule reaction can occur both in the solution inside the ESI‐charged droplets and in the gas‐phase environment of most other atmospheric pressure ionization techniques. The dimerization reaction is inherent to the electrolytic process during ESI, whereas it is more by ion/molecule chemistry in nature during APCI and APPI. From the tandem mass spectrometry (MS/MS) data, accurate mass measurements, hydrogen/deuterium (H/D) exchange experiments and density functional theory (DFT) calculations, two methyl sulfonium ions appear to be the most likely products of this electrophilic aromatic substitution reaction. The possible occurrence of this unexpected reaction complicates mass spectral data interpretation and can be misleading in terms of structural assignment as reported herein for 4‐(methyl mercapto)‐phenol. Copyright © 2009 John Wiley & Sons, Ltd.     

Structural characterization of methionine and leucine enkephalins by hydrogen/deuterium exchange and electrospray ionization tandem mass spectrometry

Conformational changes in two endogenous opioid active pentapeptides methionine enkephalin (Met-enk) and leucine enkephalin (Leu-enk) induced by trifluoroethanol (TFE) were identified using hydrogen/deuterium exchange (HDX), coupled with electrospray ionization (ESI) mass spectrometry. The exchange features in individual amino acid residues were characterized by acquiring tandem mass spectra of the deuterated peptides. The exact identity of the labile hydrogens involved in HDX reveals that the monomer forms of both peptides adopt an unfolded conformation in aqueous solvent, but prefer the 5-->2 beta-turn secondary structure under the membrane-mimetic environment. The ESI mass spectra of Met-enk and Leu-enk also reveal that the dimer structure of these peptides coexists with the monomer conformation. The extent of the dimer structure is dependent on the peptide concentration and nature of the solvent. The non-polar solvents facilitate the dimer formation.     

Study of fragmentation pattern and adsorption of 9-O-(triphenylsilyl)-10,11-dihydrocinchonidine on platinum by hydrogen/deuterium exchange using electrospray ionization ion-trap tandem mass spectrometry

We have studied the adsorption on a platinum (Pt) catalyst of two compounds utilizable as a chiral basic catalyst and a chiral modifier, dihydrocinchonidine (DHCD), and a new cinchona alkaloid derivative containing a bulky group, the Ph3SiO-DHCD molecule. The method of choice was the detection by electrospray ionization (ESI) ion-trap tandem mass spectrometry (MS/MS) of hydrogen/deuterium (H/D) exchange at room temperature, in tetrahydrofuran, at a D2 pressure of 1 bar. Based on the ESI-MS/MS spectrum of the new compound, we propose a mechanism for the formation of the silatropylium cation containing a Si-O bond. From the fragmentation pattern of Ph3SiO-DHCD it was confirmed that ESI-ion-trap MS/MS can be used to study the adsorption processes of complicated carbon compounds by investigating their H/D exchange reactions. In the case of Ph3SiO-DHCD, the results demonstrate that H/D exchange takes place mainly on the quinoline skeleton. However, the strong pi-bonded adsorption of the quinoline skeleton parallel with the imaginary plane of Pt is not preferred because the bulky Ph3Si group inhibits the multiple pi-bonded adsorption of the Ph3SiO-DHCD. Because of this hindrance the molecule was adsorbed tilted via the nonbonding electron pair of the N atom and C2' atom of the quinoline skeleton; consequently, mainly alkaloid-d1 and alkaloid-d2 are formed.     

'On-the-fly' hydrogen/deuterium exchange liquid chromatography/mass spectrometry using a dual-sprayer atmospheric pressure ionisation source

In-source 'on-the-fly' hydrogen/deuterium (H/D) exchange liquid chromatography mass spectrometry (LC/MS) has been investigated. The work was performed using a dual-sprayer source. The analyte was introduced through an electrospray ionisation sprayer and D2O was introduced through an atmospheric pressure chemical ionisation sprayer. To achieve H/D exchange sufficient to determine the number of exchangeable H atoms of a compound, a saturated 'steady-state' D2O atmosphere had to be created in the ion source by having a 2:1 or higher D2O-to-analyte flow rate ratio. Under these conditions H/D exchange levels of 32-90% were achieved. In most cases the H/D exchange was sufficient to measure the number of exchangeable H atoms in some antiulcerative and anthelmintic pharmaceuticals. The concept of in-source 'on-the-fly' H/D exchange by introducing the deuterating agent via a second sprayer has been shown. It allows the integrity of the chromatographic separation to be kept, since the H/D exchange takes place post-separation.     

Hydrogen/deuterium exchange on aromatic rings during atmospheric pressure chemical ionization mass spectrometry

It has been demonstrated that substituted indoles fully labelled with deuterium on the aromatic ring can undergo substantial exchange back to partial and even fully protonated forms during atmospheric pressure chemical ionisation (APCI) liquid chromatography/mass spectrometry (LC/MS). The degree of this exchange was strongly dependent on the absolute quantity of analyte, the APCI desolvation temperature, the nature of the mobile phase, the mobile phase flow rate and the instrument used. Hydrogen/deuterium (H/D) exchange on several other aromatic ring systems during APCI LC/MS was either undetectable (nitrobenzene, aniline) or extremely small (acetanilide) compared to the effect observed for substituted indoles. This observation has major implications for quantitative assays using deuterium‐labelled internal standards and for the detection of deuterium‐labelled products from isotopically labelled feeding experiments where there is a risk of back exchange to the protonated form during the analysis. Copyright © 2010 John Wiley & Sons, Ltd.     

Multidimensional separations of ubiquitin conformers in the gas phase: Relating ion cross sections to H/D exchange measurements

Investigating gas-phase structures of protein ions can lead to an improved understanding of intramolecular forces that play an important role in protein folding. Both hydrogen/deuterium (H/D) exchange and ion mobility spectrometry provide insight into the structures and stabilities of different gas-phase conformers, but how best to relate the results from these two methods has been hotly debated. Here, high-field asymmetric waveform ion mobility spectrometry (FAIMS) is combined with Fourier-transform ion cyclotron resonance mass spectrometry (FT/ICR MS) and is used to directly relate ubiquitin ion cross sections and H/D exchange extents. Multiple conformers can be identified using both methods. For the 9+ charge state of ubiquitin, two conformers (or unresolved populations of conformers) that have cross sections differing by 10% are resolved by FAIMS, but only one conformer is apparent using H/D exchange at short times. For the 12+ charge state, two conformers (or conformer populations) have cross sections differing by <1%, yet H/D exchange of these conformers differ significantly (6 versus 25 exchanges). These and other results show that ubiquitin ion collisional cross sections and H/D exchange distributions are not strongly correlated and that factors other than surface accessibility appear to play a significant role in determining rates and extents of H/D exchange. Conformers that are not resolved by one method could be resolved by the other, indicating that these two methods are highly complementary and that more conformations can be resolved with this combination of methods than by either method alone.     

Detection of oligonucleotide gas-phase conformers: H/D exchange and ion mobility as complementary techniques

Gas-phase hydrogen/deuterium exchange of small oligonucleotides (dTG, dC(6) and C(6)) with CD(3)OD was performed in the second hexapole of a Fourier transform ion-cyclotron resonance (FTICR) mass spectrometer. Ion activation experiments were conducted by accelerating the ions at the entrance of the H/D exchange cell under conditions promoting exclusively collisional isomerization. These experiments allowed us to assess the presence of several conformers, and to probe the height of the isomerization barrier separating these conformers. Ion mobility experiments were also performed. Their results were consistent with the H/D exchange data. A model accounting for the competing isomerization and H/D exchange reactions is proposed. Comparing the ion acceleration experiments for H/D exchange and for ion mobility reveals that the most compact conformer displays the fastest H/D exchange. This observation shows that H/D exchange and ion mobility provide us with complementary information because hydrogen accessibility and macromolecule compactness are not univocally associated.     

Conformational changes in beta-endorphin as studied by electrospray ionization mass spectrometry.

Because of a wide range of physiological functions, the structure of beta-endorphin (BE) is of great interest. In this study, conformational changes in BE induced by methanol are explored with electrospray ionization-mass spectrometry (ESI-MS). Differences in the charge-state distribution (CSD) and the extent of hydrogen/deuterium (H/D) exchange were used to monitor the conformational changes. The latter experiments were conducted via time-resolved ESI-MS in a continuous-flow apparatus. Both these techniques demonstrate that BE exists in a random coil open structure in aqueous media, but it acquires a more compact conformation with increased concentration of methanol. The H/D exchange experiments reveal that BE forms 61% alpha-helix in mixed solvents.     

A gated-beam electrospray ionization source with an external ion reservoir. A new tool for the characterization of biomolecules using electrospray ionization mass spectrometry.

In this work we describe a micro-electrospray ionization source equipped with an atmospheric pressure external ion shutter. The solenoid-activated shutter prevents the electrospray plume from entering the inlet capillary unless triggered to the 'open' position. When in the 'closed' position, a stable electrospray plume is maintained between the electrospray ionization (ESI) emitter and the electrically isolated face of the shutter. When the shutter is triggered, a 'slice' of ions is allowed to enter the inlet capillary and is subsequently accumulated in an external ion reservoir comprised of a radio frequency only (rf-only) hexapole and a pair of electrostatic elements. Following ion accumulation in the external ion reservoir, intact molecular ions of proteins, oligonucleotides, and noncovalent complexes can be stored for extended intervals (>30 minutes) prior to being transferred to the Fourier transform ion cyclotron resonance (FTICR) trapped ion cell for mass analysis. By introducing reactive gases directly into the external ion reservoir during the storage interval, ion-molecule reactions, such as H/D exchange, can be performed at high effective pressures. This scheme obviates the need for the long reaction times and delays associated with restoring base pressure in the trapped ion cell and allows H/D exchange reactions to be conducted in a fraction of the time required using conventional in-cell exchange approaches. The back face of the shutter arm contains an elastomeric material which can be positioned to seal the inlet to the mass spectrometer resulting in lower base pressure in the ion reservoir and the FTICR cell. Additionally, it is noted that blocking the ESI plume during non-accumulation events results in reduced fouling of the source electrodes and longer times between required source cleaning.     

Hydrogen/deuterium exchange using a coaxial sheath-flow interface for capillary electrophoresis/mass spectrometry

The interfacing of capillary electrophoresis (CE) with mass spectrometry (MS) is well established and may be accomplished by use of either a coaxial arrangement or by employing a liquid T-junction. In both these interfaces a make-up flow is introduced. This is required because of the mismatch in flow rates for capillary electrophoresis approximately nL/min and 'true' electrospray approximately 2-10 microL/min. Electrical connectivity may also be established where the liquid flows meet (the introduction of nanospray renders the use of make-up flow unnecessary). Hydrogen/deuterium (H/D) exchange occurs in solution when there are labile hydrogen atoms present in a molecule. The establishment of the presence and the number of such exchangeable hydrogen atoms may be of importance in the identification and differentiation of compounds. It may also be an aid in the structural elucidation of unknown materials. We have investigated the feasibility of carrying out H/D exchange via a CE/MS interface. This involved the addition of D2O to the sheath flow and our preliminary results showing the separations of drug substances, subsequently undergoing exchange, are presented.