59
Y. Kostyukevich et al., Eur. J. Mass Spectrom. 21, 59–63 (2015) Received: 31 March 2015 n Revised: 8 April 2015 n Accepted: 8 April 2015 n Publication: 21 April 2015
EUROPEAN JOURNAL OF MASS SPECTROMETRY
Letter
Analytical potential of the in-electrospray ionization source hydrogen/deuterium exchange for the investigation of oligonucleotides Yury Kostyukevich,a,b,c Alexey Kononikhin,b,c Igor Popov,c,d Natalia Starodubtzeva,c,e Stanislav Pekov,b Eugene Kukaev,c,d Maria Indeykinab and Eugene Nikolaeva,b,c a
Skolkovo Institute of Science and Technology, Novaya St. 100, Skolkovo 143025, Russian Federation. E-mail: [email protected]
b
Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Leninskij pr. 38 k.2, 119334 Moscow, Russian Federation
c
Moscow Institute of Physics and Technology, 141700 Dolgoprudnyi, Moscow Region, Russian Federation
d
Emanuel Institute for Biochemical Physic,s Russian Academy of Sciences, Kosygina St. 4, 119334 Moscow, Russian Federation
e
Research Center for Obstetrics, Gynecology and Perinatology, 4 Oparin St., Moscow 117997, Russian Federation It has previously been reported that different conformations of oligonucleotides may be detected using a gas-phase hydrogen/deuterium (H/D) exchange performed in the collision cell of a mass spectrometer. The presence of different conformers was postulated based on the bimodal shape of the deuterium distribution and on the ion mobility spectrometry data. Here we implement an in-electrospray ionization source H/D exchange to detect the different conformations of oligonucleotides in the region of ion formation. We observed that the number of H/D exchanges depends considerably on the temperature of the desolvating capillary and varies from 25% at 50°C to 80% at 450°C, but no bimodality in the shape of the deuterium distribution was observed. Such results indicate that in the region of ion formation different conformations of oligonucleotide ions rapidly interconvert one into another.
Keywords: oligonucleotides, H/D exchange, mass spectrometry, ESI, FT ICR
Introduction The investigation of the conformations of biomolecules is an important challenge in the analytical chemistry and hydrogen/ deuterium (H/D) exchange mass spectrometry (MS) is one of the most important methods for the investigation of conformers of neutral molecules and gas-phase ions. Liquidphase H/D exchange is the routine approach for the investigation of protein conformation dynamics and the determination of binding sites of large biomolecules.1,2 Gas-phase ISSN: 1469-0667 doi: 10.1255/ejms.1330
H/D exchange performed in the high-vacuum part of the mass spectrometer was used to investigate the structure and gaseous conformations of peptides,3 proteins,4,5 oligosaccharides,6 oligonucleotides7,8 and other molecules.9 The disadvantages of this approach is the long-lasting post-experimental cleaning of the equipment, because the ultimate removal of traces of deuterated gas (such as D2O, ND3, MeOD etc.) from the high-vacuum region requires a long time. Another limita© IM Publications LLP 2015 All rights reserved
60
Potential of the in-ESI Source Hydrogen/Deuterium Exchange in Oligonucleotide Investigations
tion is that such experiments can be performed only on mass spectrometers equipped with a collision cell. Previously, it was proposed that the H/D exchange be performed directly in the ionization source.10,11 Such an approach makes it possible to obtain the high deuteration level for peptides,12 proteins,13,14 oligosaccharides,15 and low-weight organic molecules.16–20 However, the investigation of the oligonucleotides using an in-ionization source H/D exchange has not been performed yet. In this paper we demonstrate the potential of the in-electrospray ionization (ESI) source H/D exchange for the investigation of the gas-phase ions of oligonucleotides.
Methods
Sample preparation
The oligonucleotide (dA 20) was purchased from Evrogen (Moscow, Russia) and was prepared in the 1:1 mixture of water and methanol at a concentration of 10–5 M. All chemicals were of analytical grade.
MS analysis All the experiments were performed on a LTQ FT Ultra (Thermo Electron Corp., Bremen, Germany) mass spectrometer equipped with a 7T superconducting magnet. Ions were generated by an IonMax electrospray ion source (Thermo Electron Corp., Bremen, Germany) in a negative ESI mode. The temperature of the desolvating capillary was varied from 50°C to 450°C. The length of the desolvating capillary was 105 mm and its inner diameter was 0.5 mm. The infusion rate of the sample was 1 µL min–1 and the needle voltage was 3000 V.
In-ESI source H/D exchange To create an atmosphere saturated with D2O vapors, 400 µL of solvent were placed on a copper plate positioned at approximately 7 mm below the ESI needle (see Figure 1). Owing to the evaporation of the droplet an atmosphere of the saturated D2O
Figure 1. The design of the in-ESI source H/D exchange experiment. The temperature of the desolvating capillary was varied from 50°C to 450°C.
vapors was created in the region between the ESI needle and the MS entrance capillary. The ESI needle was 5 mm from the desolvating capillary inlet.
Results and discussions The structure of oligonucleotide ions in the gas phase was intensively studied using ion mass spectrometry (IMS), various labelling techniques, including H/D exchange, ion spectroscopy and computer simulation.21 Mainly, the researchers were interested in the preserving22 or unfolding23 of the hairpin structure during the ionization. Here we are trying to implement the simple and cheap in-ESI source H/D exchange approach to investigate the oligonucleotides conformations in the gas phase in the region of ion formation. The design of the experimental setup for performing the H/D exchange reaction is presented in Figure 1. Charged droplets are produced by the ESI source, passed through the desolvating capillary and evaporate to produce the gas-phase ions. During the transport the gas-phase ions interact with the vapors of the D2O and replace their labile hydrogens with deuteriums. We have implemented the in-ESI source H/D exchange to investigate the model oligonucleotide dA20, which does not form a hairpin structure. The structure of the dA20 is presented in Figure 2(a); this oligonucleotide has 19 OH groups from phosphate residues, two terminus OH groups and 20 NH2 groups. All the hydrogen atoms are labile and can exchange for deuterium under our experimental conditions. Our goal was to measure the level of deuteration as a function of the desolvating capillary. It was observed that for low temperatures of the desolvating capillary, low-charge states form clusters with the solvent [Figure 2(b)]. High-charge states almost do not form such clusters. The results of the H/D exchange experiment are presented in the Figure 3. It can be seen that all the charge states undergo H/D exchange and the level of deuteration increases with the temperature of the desolvating capillary. It is also clear that for all temperatures in the range 50°C to 450°C the deuterium distribution remains monomodal. Previously, Balbeur et al.7 performed a H/D exchange reaction with oligonucleotides in a collisional cell and observed that the deuterium distribution became bimodal. For example, when ion [C6 – 2H]2– was allowed to collide with CD3OD at a pressure of 10–3 mbar for 2.5 s the bimodal deuterium distribution with peaks corresponding to 7 and 15 exchanges was observed. Such results indicate the presence of two different conformations of oligonucleotide gas-phase ions: one is folded, so functional groups are not easily accessible for deuterium, and another is unfolded. Also Gidden et al.24 investigated the gas-phase structure of [dTG – H]– and [dGT – H]– and observed that under 80 K the IMS data showed a bimodal arrival time distribution, while at 300 K it became monomodal. Such results indicate that at 300 K, the average energy in the system is well above the isomerization barrier and two conformers rapidly inter-
Y. Kostyukevich et al., Eur. J. Mass Spectrom. 21, 59–63 (2015) 61
Figure 2. (a) The structure of the investigated dA20 oligonucleotide. (b) The mass spectra of the dA20 obtained at different temperatures of the desolvating capillary. Functional groups, containing labile hydrogens, are color coded: blue, violet, OH; red, NH2.
convert. The bimodal and multimodal shape of the deuterium distribution was also observed many times during the gasphase H/D exchange experiments of proteins,25 which indicates that different charge states of proteins adopt different mixtures of conformations.4,5,26 Also, a bimodal deuterium distribution was observed for the oligosaccharides.15 Our results with oligonucleotides show that the shape of the deuterium distribution remains monomodal. This means that in the region of ion formation, different conformations of oligonucleotide ions rapidly interconvert one into another,
Figure 3. The H/D exchange of the different charge states of the dA20 at different temperatures of the desolvating capillary. (a) Full spectrum, (b) the –6 charge state and (c) the –4 charge state. The symbol S indicates the solvent adducts at a low temperature of the desolvating capillary.
because they are relatively “hot” and have sufficient internal energy to do so. There is also a possibility that different charge states of dA20 could correspond to different conformations. Indeed, it is known that the different charge states of proteins correspond to different conformations and the more unfolded the conformation is, the higher the charge states observed.27,28 The results of the H/D exchange for the different charge states of dA20 as a function of the temperature of the desolvating capillary are presented in the Figure 4. It can be seen that all the charge states demonstrate an almost similar level of H/D exchange, which indicates the absence of differently folded gas-phase ions. At the lowest temperature of 50°C, dA20 demonstrate ~15 H/D exchanges, which is close to the number of OH groups in the molecule. With an increase of the temperature to 200°C the number of exchanges slowly increases to ~20, which is almost equal to the number of OH groups, and with a subsequent increase of temperature the number of exchanges increases much more rapidly. This means that under elevated temperatures the NH2 groups also participate in the H/D exchange reaction.
Figure 4. The dependence of the H/D exchange level on the temperature of the desolvating capillary. Colors correspond to the different charge states.
62
Potential of the in-ESI Source Hydrogen/Deuterium Exchange in Oligonucleotide Investigations
So we have demonstrated the analytical potential of an in-ESI source H/D exchange approach to the investigation of oligonucleotides. The method is simple, robust, provides a high level of deuteration and can be implemented with any commercial mass spectrometer.
Author contributions All the authors contributed to writing the manuscript. All authors have given approval to the final version of the manuscript
Acknowledgments This work was supported by the Russian Scientific Foundation (grant 14-24-00114).
Competing financial interest The authors declare no competing financial interests.
References 1 V. Katta and B.T. Chait, “Hydrogen-deuterium exchange
2
3
4
5
6
electrospray-ionization mass-spectrometry—a method for probing protein conformational-changes in solution”, J. Am. Chem. Soc. 115, 6317 (1993). doi: http://dx.doi. org/10.1021/ja00067a054 T.E. Wales and J.R. Engen, “Hydrogen exchange mass spectrometry for the analysis of protein dynamics”, Mass Spectrom. Rev. 25, 158 (2006). doi: http://dx.doi. org/10.1002/mas.20064 E. Gard, M.K. Green, J. Bregar and C.B. Lebrilla, “Gasphase hydrogen–deuterium exchange as a molecular probe for the interaction of methanol and protonated peptides”, J. Am. Chem. Soc. Mass Spectrom. 5, 623 (1994). doi: http://dx.doi.org/10.1016/1044-0305(94)85003-8 F.W. McLafferty, Z.Q. Guan, U. Haupts, T.D. Wood and N.L. Kelleher, “Gaseous conformational structures of cytochrome C”, J. Am. Chem. Soc. 120, 4732 (1998). doi: http://dx.doi.org/10.1021/ja9728076 M.A. Freitas, C.L. Hendrickson, M.R. Emmett and A.G. Marshall, “Gas-phase bovine ubiquitin cation conformations resolved by gas-phase hydrogen/deuterium exchange rate and extent”, Int. J. Mass Spectrom. 185, 565 (1999). doi: http://dx.doi.org/10.1016/S13873806(98)14172-6 N.P.J. Price, “Oligosaccharide structures studied by hydrogen–deuterium exchange and MALDI-TOF mass spectrometry”, Anal. Chem. 78, 5302 (2006). doi: http:// dx.doi.org/10.1021/ac052168e
7 D. Balbeur, J. Widart, B. Leyh, L. Cravello and E. De
Pauw, “Detection of oligonucleotide gas-phase conformers: H/D exchange and ion mobility as complementary techniques”, J. Am. Chem. Soc. Mass Spectrom. 19, 938 (2008). doi: http://dx.doi.org/10.1016/j.jasms.2008.03.018 8 K.B. Green-Church, P.A. Limbach, M.A. Freitas and A.G. Marshall, “Gas-phase hydrogen/deuterium exchange of positively charged mononucleotides by use of Fouriertransform ion cyclotron resonance mass spectrometry”, J. Am. Chem. Soc. Mass Spectrom. 12, 268 (2001). doi: http://dx.doi.org/10.1016/S1044-0305(00)00222-1 9 T. Solouki, M.A. Freitas and A. Alomary, “Gas-phase hydrogen/deuterium exchange reactions of fulvic acids: an electrospray ionization Fourier transform ion cyclotron resonance mass spectral study”, Anal. Chem. 71, 4719 (1999). doi: http://dx.doi.org/10.1021/ac990185w 10 Y. Kostyukevich, A. Kononikhin, I. Popov and E. Nikolaev, “Simple atmospheric hydrogen/deuterium exchange method for enumeration of labile hydrogens by electrospray ionization mass spectrometry”, Anal. Chem. 85, 5330 (2013). doi: http://dx.doi.org/10.1021/ac4006606 11 M.E. Hemling, J.J. Conboy, M.F. Bean, M. Mentzer and S.A. Carr, “Gas-phase hydrogen-deuterium exchange in electrospray-ionization mass-spectrometry as a practical tool for structure elucidation”, J. Am. Chem. Soc. Mass Spectrom. 5, 434 (1994). doi: http://dx.doi. org/10.1016/1044-0305(94)85059-3 12 Z. Takats, S.C. Nanita, G. Schlosser, K. Vekey and R.G. Cooks, “Atmospheric pressure gas-phase H/D exchange of serine octamers”, Anal. Chem. 75, 6147 (2003). doi: http://dx.doi.org/10.1021/ac034284s 13 Y. Kostyukevich, A. Kononikhin, I. Popov and E. Nikolaev, “Conformational changes of ubiquitin during electrospray ionization as determined by in-ESI source H/D exchange combined with high-resolution MS and ECD fragmentation”, J. Mass Spectrom. 49, 989 (2014). doi: http://dx.doi. org/10.1002/jms.3409 14 Y. Kostyukevich, A. Kononikhin, I. Popov, A. Spasskiy and E. Nikolaev, “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”, J. Mass Spectrom. 50, 49 (2015). doi: http:// dx.doi.org/10.1002/jms.3535 15 Y. Kostyukevich, A. Kononikhin, I. Popov and E. Nikolaev, “In-ESI source hydrogen/deuterium exchange of carbohydrate ions”, Anal. Chem. 86, 2595 (2014). doi: http:// dx.doi.org/10.1021/ac4038202 16 Y. Kostyukevich, A. Kononikhin, I. Popov, O. Kharybin, I. Perminova, A. Konstantinov and E. Nikolaev, “Enumeration of labile hydrogens in natural organic matter by use of hydrogen/deuterium exchange Fourier transform ion cyclotron resonance mass spectrometry”, Anal. Chem. 85, 11007 (2013). doi: http://dx.doi. org/10.1021/ac402609x
Y. Kostyukevich et al., Eur. J. Mass Spectrom. 21, 59–63 (2015) 63
17 Y. Kostyukevich, A. Kononikhin, I. Popov, N.
23 A. Arcella, J. Dreyer, E. Ippoliti, I. Ivani, G. Portella,
Starodubtseva, E. Kukaev and E. Nikolaev, “Letter: Separation of tautomeric forms of [2-nitrophloroglucinol-H] – by an in-electrospray ionization source hydrogen/deuterium exchange approach”, Eur. J. Mass Spectrom. 20, 345 (2014). doi: http://dx.doi.org/10.1255/ ejms.1282 18 Y. Kostyukevich, A. Kononikhin, A. Zherebker, I. Popov, I. Perminova and E. Nikolaev, “Enumeration of non-labile oxygen atoms in dissolved organic matter by use of 16 O/18O exchange and Fourier transform ion-cyclotron resonance mass spectrometry”, Anal. Bioanal. Chem. 406, 6655 (2014). doi: http://dx.doi.org/10.1007/s00216014-8097-9 19 A. Ahmed and S. Kim, “Atmospheric pressure photo ionization hydrogen/deuterium exchange mass spectrometry—a method to differentiate isomers by mass spectrometry”, J. Am. Chem. Soc. Mass Spectrom. 24, 1900 (2013). doi: http://dx.doi.org/10.1007/s13361-013-0726-6 20 Y. Cho, A. Ahmed and S. Kim, “Application of atmospheric pressure photo ionization hydrogen/deuterium exchange high-resolution mass spectrometry for the molecular level speciation of nitrogen compounds in heavy crude oils”, Anal. Chem. 85, 9758 (2013). doi: http://dx.doi.org/10.1021/ac402157r 21 J. Abi-Ghanem and V. Gabelica, “Nucleic acid ion structures in the gas phase”, Phys. Chem. Chem. Phys. 16, 21204 (2014). doi: http://dx.doi.org/10.1039/C4CP02362E 22 J.J.E. Mo, G.C. Todd and K. Hakansson, “Characterization of nucleic acid higher order structure by gas-phase H/D exchange in a quadrupole-FT-ICR mass spectrometer”, Biopolymers 91, 256 (2009). doi: http://dx.doi.org/10.1002/bip.21134
V. Gabelica, P. Carloni and M. Orozco, “Structure and dynamics of oligonucleotides in the gas phase”, Angew. Chem. Int. Ed. 54, 467 (2015). doi: http://dx.doi. org/10.1002/ange.201406910 24 J. Gidden, J.E. Bushnell and M.T. Bowers, “Gas-phase conformations and folding energetics of oligonucleotides: dTG – and dGT–”, J. Am. Chem. Soc. 123, 5610 (2001). doi: http://dx.doi.org/10.1021/ja015940d 25 B.C. Bohrer, N. Atlasevich and D.E. Clemmer, “Transitions between elongated conformations of ubiquitin [M + 11H]11+ enhance hydrogen/deuterium exchange”, J. Phys. Chem. B 115, 4509 (2011). doi: http://dx.doi. org/10.1021/jp2008495 26 M. Nousiainen, P. Vainiotalo, P.J. Derrick, H.J. Cooper, A. Hoxha, D. Fati, H.R. Trayer, D.G. Ward and I.P. Trayer, “Calcium and peptide binding to folded and unfolded conformations of cardiac Troponin C. Electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry”, Eur. J. Mass Spectrom. 8, 471 (2002). doi: http://dx.doi.org/10.1255/ejms.523 27 A. Kharlamova, J.C. DeMuth and S.A. McLuckey, “Vapor treatment of electrospray droplets: evidence for the folding of initially denatured proteins on the sub-millisecond time-scale”, J. Am. Chem. Soc. Mass Spectrom. 23, 88 (2012). doi: http://dx.doi.org/10.1007/s13361-0110258-x 28 E.W. Robinson and E.R. Williams, “Multidimensional separations of ubiquitin conformers in the gas phase: relating ion cross sections to H/D exchange measurements”, J. Am. Chem. Soc. Mass Spectrom. 16, 1427 (2005). doi: http://dx.doi.org/10.1016/j.jasms.2005.04.007
Y. Kostyukevich et al., Eur. J. Mass Spectrom. 21, 59–63 (2015) Received: 31 March 2015 n Revised: 8 April 2015 n Accepted: 8 April 2015 n Publication: 21 April 2015
EUROPEAN JOURNAL OF MASS SPECTROMETRY
Letter
Analytical potential of the in-electrospray ionization source hydrogen/deuterium exchange for the investigation of oligonucleotides Yury Kostyukevich,a,b,c Alexey Kononikhin,b,c Igor Popov,c,d Natalia Starodubtzeva,c,e Stanislav Pekov,b Eugene Kukaev,c,d Maria Indeykinab and Eugene Nikolaeva,b,c a
Skolkovo Institute of Science and Technology, Novaya St. 100, Skolkovo 143025, Russian Federation. E-mail: [email protected]
b
Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Leninskij pr. 38 k.2, 119334 Moscow, Russian Federation
c
Moscow Institute of Physics and Technology, 141700 Dolgoprudnyi, Moscow Region, Russian Federation
d
Emanuel Institute for Biochemical Physic,s Russian Academy of Sciences, Kosygina St. 4, 119334 Moscow, Russian Federation
e
Research Center for Obstetrics, Gynecology and Perinatology, 4 Oparin St., Moscow 117997, Russian Federation It has previously been reported that different conformations of oligonucleotides may be detected using a gas-phase hydrogen/deuterium (H/D) exchange performed in the collision cell of a mass spectrometer. The presence of different conformers was postulated based on the bimodal shape of the deuterium distribution and on the ion mobility spectrometry data. Here we implement an in-electrospray ionization source H/D exchange to detect the different conformations of oligonucleotides in the region of ion formation. We observed that the number of H/D exchanges depends considerably on the temperature of the desolvating capillary and varies from 25% at 50°C to 80% at 450°C, but no bimodality in the shape of the deuterium distribution was observed. Such results indicate that in the region of ion formation different conformations of oligonucleotide ions rapidly interconvert one into another.
Keywords: oligonucleotides, H/D exchange, mass spectrometry, ESI, FT ICR
Introduction The investigation of the conformations of biomolecules is an important challenge in the analytical chemistry and hydrogen/ deuterium (H/D) exchange mass spectrometry (MS) is one of the most important methods for the investigation of conformers of neutral molecules and gas-phase ions. Liquidphase H/D exchange is the routine approach for the investigation of protein conformation dynamics and the determination of binding sites of large biomolecules.1,2 Gas-phase ISSN: 1469-0667 doi: 10.1255/ejms.1330
H/D exchange performed in the high-vacuum part of the mass spectrometer was used to investigate the structure and gaseous conformations of peptides,3 proteins,4,5 oligosaccharides,6 oligonucleotides7,8 and other molecules.9 The disadvantages of this approach is the long-lasting post-experimental cleaning of the equipment, because the ultimate removal of traces of deuterated gas (such as D2O, ND3, MeOD etc.) from the high-vacuum region requires a long time. Another limita© IM Publications LLP 2015 All rights reserved
60
Potential of the in-ESI Source Hydrogen/Deuterium Exchange in Oligonucleotide Investigations
tion is that such experiments can be performed only on mass spectrometers equipped with a collision cell. Previously, it was proposed that the H/D exchange be performed directly in the ionization source.10,11 Such an approach makes it possible to obtain the high deuteration level for peptides,12 proteins,13,14 oligosaccharides,15 and low-weight organic molecules.16–20 However, the investigation of the oligonucleotides using an in-ionization source H/D exchange has not been performed yet. In this paper we demonstrate the potential of the in-electrospray ionization (ESI) source H/D exchange for the investigation of the gas-phase ions of oligonucleotides.
Methods
Sample preparation
The oligonucleotide (dA 20) was purchased from Evrogen (Moscow, Russia) and was prepared in the 1:1 mixture of water and methanol at a concentration of 10–5 M. All chemicals were of analytical grade.
MS analysis All the experiments were performed on a LTQ FT Ultra (Thermo Electron Corp., Bremen, Germany) mass spectrometer equipped with a 7T superconducting magnet. Ions were generated by an IonMax electrospray ion source (Thermo Electron Corp., Bremen, Germany) in a negative ESI mode. The temperature of the desolvating capillary was varied from 50°C to 450°C. The length of the desolvating capillary was 105 mm and its inner diameter was 0.5 mm. The infusion rate of the sample was 1 µL min–1 and the needle voltage was 3000 V.
In-ESI source H/D exchange To create an atmosphere saturated with D2O vapors, 400 µL of solvent were placed on a copper plate positioned at approximately 7 mm below the ESI needle (see Figure 1). Owing to the evaporation of the droplet an atmosphere of the saturated D2O
Figure 1. The design of the in-ESI source H/D exchange experiment. The temperature of the desolvating capillary was varied from 50°C to 450°C.
vapors was created in the region between the ESI needle and the MS entrance capillary. The ESI needle was 5 mm from the desolvating capillary inlet.
Results and discussions The structure of oligonucleotide ions in the gas phase was intensively studied using ion mass spectrometry (IMS), various labelling techniques, including H/D exchange, ion spectroscopy and computer simulation.21 Mainly, the researchers were interested in the preserving22 or unfolding23 of the hairpin structure during the ionization. Here we are trying to implement the simple and cheap in-ESI source H/D exchange approach to investigate the oligonucleotides conformations in the gas phase in the region of ion formation. The design of the experimental setup for performing the H/D exchange reaction is presented in Figure 1. Charged droplets are produced by the ESI source, passed through the desolvating capillary and evaporate to produce the gas-phase ions. During the transport the gas-phase ions interact with the vapors of the D2O and replace their labile hydrogens with deuteriums. We have implemented the in-ESI source H/D exchange to investigate the model oligonucleotide dA20, which does not form a hairpin structure. The structure of the dA20 is presented in Figure 2(a); this oligonucleotide has 19 OH groups from phosphate residues, two terminus OH groups and 20 NH2 groups. All the hydrogen atoms are labile and can exchange for deuterium under our experimental conditions. Our goal was to measure the level of deuteration as a function of the desolvating capillary. It was observed that for low temperatures of the desolvating capillary, low-charge states form clusters with the solvent [Figure 2(b)]. High-charge states almost do not form such clusters. The results of the H/D exchange experiment are presented in the Figure 3. It can be seen that all the charge states undergo H/D exchange and the level of deuteration increases with the temperature of the desolvating capillary. It is also clear that for all temperatures in the range 50°C to 450°C the deuterium distribution remains monomodal. Previously, Balbeur et al.7 performed a H/D exchange reaction with oligonucleotides in a collisional cell and observed that the deuterium distribution became bimodal. For example, when ion [C6 – 2H]2– was allowed to collide with CD3OD at a pressure of 10–3 mbar for 2.5 s the bimodal deuterium distribution with peaks corresponding to 7 and 15 exchanges was observed. Such results indicate the presence of two different conformations of oligonucleotide gas-phase ions: one is folded, so functional groups are not easily accessible for deuterium, and another is unfolded. Also Gidden et al.24 investigated the gas-phase structure of [dTG – H]– and [dGT – H]– and observed that under 80 K the IMS data showed a bimodal arrival time distribution, while at 300 K it became monomodal. Such results indicate that at 300 K, the average energy in the system is well above the isomerization barrier and two conformers rapidly inter-
Y. Kostyukevich et al., Eur. J. Mass Spectrom. 21, 59–63 (2015) 61
Figure 2. (a) The structure of the investigated dA20 oligonucleotide. (b) The mass spectra of the dA20 obtained at different temperatures of the desolvating capillary. Functional groups, containing labile hydrogens, are color coded: blue, violet, OH; red, NH2.
convert. The bimodal and multimodal shape of the deuterium distribution was also observed many times during the gasphase H/D exchange experiments of proteins,25 which indicates that different charge states of proteins adopt different mixtures of conformations.4,5,26 Also, a bimodal deuterium distribution was observed for the oligosaccharides.15 Our results with oligonucleotides show that the shape of the deuterium distribution remains monomodal. This means that in the region of ion formation, different conformations of oligonucleotide ions rapidly interconvert one into another,
Figure 3. The H/D exchange of the different charge states of the dA20 at different temperatures of the desolvating capillary. (a) Full spectrum, (b) the –6 charge state and (c) the –4 charge state. The symbol S indicates the solvent adducts at a low temperature of the desolvating capillary.
because they are relatively “hot” and have sufficient internal energy to do so. There is also a possibility that different charge states of dA20 could correspond to different conformations. Indeed, it is known that the different charge states of proteins correspond to different conformations and the more unfolded the conformation is, the higher the charge states observed.27,28 The results of the H/D exchange for the different charge states of dA20 as a function of the temperature of the desolvating capillary are presented in the Figure 4. It can be seen that all the charge states demonstrate an almost similar level of H/D exchange, which indicates the absence of differently folded gas-phase ions. At the lowest temperature of 50°C, dA20 demonstrate ~15 H/D exchanges, which is close to the number of OH groups in the molecule. With an increase of the temperature to 200°C the number of exchanges slowly increases to ~20, which is almost equal to the number of OH groups, and with a subsequent increase of temperature the number of exchanges increases much more rapidly. This means that under elevated temperatures the NH2 groups also participate in the H/D exchange reaction.
Figure 4. The dependence of the H/D exchange level on the temperature of the desolvating capillary. Colors correspond to the different charge states.
62
Potential of the in-ESI Source Hydrogen/Deuterium Exchange in Oligonucleotide Investigations
So we have demonstrated the analytical potential of an in-ESI source H/D exchange approach to the investigation of oligonucleotides. The method is simple, robust, provides a high level of deuteration and can be implemented with any commercial mass spectrometer.
Author contributions All the authors contributed to writing the manuscript. All authors have given approval to the final version of the manuscript
Acknowledgments This work was supported by the Russian Scientific Foundation (grant 14-24-00114).
Competing financial interest The authors declare no competing financial interests.
References 1 V. Katta and B.T. Chait, “Hydrogen-deuterium exchange
2
3
4
5
6
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