The Use of Ion Mobility Mass Spectrometry for Isomer Composition

Article pubs.acs.org/ac

The Use of Ion Mobility Mass Spectrometry for Isomer Composition Determination Extracted from Se-Rich Yeast Johann Far,† Cédric Delvaux,† Christopher Kune,† Gauthier Eppe,‡,† and Edwin de Pauw*,† †

Department of Chemistry, Laboratory of Mass Spectrometry, University of Liège, 3 Allée de la chimie, B6C, Liege (Sart Tilman), B-4000, Belgium ‡ Centre of Analytical Research and Technology (CART) - LSM/Inorganic Analytical Chemistry, Department of Chemistry, University of Liège, 3, Allee de la Chimie B6C, Liege, 4000, Belgium S Supporting Information *

ABSTRACT: The isomer ratio determination of a seleniumcontaining metabolite produced by Se-rich yeast was performed. Electrospray ionization and ion mobility mass spectrometry (IMMS) were unsuccessfully used in order to resolve the isomers according to their collisional cross section (CCS) difference. The isomer ratio determination of 2,3-dihydroxypropionylselenocystathionine was performed after multidimensional liquid chromatography preconcentration from a water extract of Se-rich yeast using preparative size exclusion, anion exchange, and capillary reverse phase columns coupled to IM-MS. 4′-nitrobenzo-15crown-5 ether, a selective shift reagent (SSR), was added after the last chromatographic dimension in order to specifically increase the CCS of one of the isomers by the formation of a stable host− guest system with the crown ether. Both isomers were consequently fully resolved by IM-MS, and the relative ratio of the isomers was determined to be 11−13% and 87−89%. The present data compared favorably with the literature to support the analytical strategy despite the lack of an authentic standard for method validation. In addition, computational chemistry methods were successfully applied to design the SSR and to support the experimental data.

S

chemopreventive agents against cancer could depend on the type of isomers provided to rat cells or rat whole body as demonstrated by Sohn and co-workers.10,11 Up to now, no general (routine) purpose analytical tool enables one to perform the isomeric ratio determination of such isomeric mixtures. In some cases, chromatographic methods such as hydrophilic interaction liquid chromatography, HILIC, could lead to isomer separation.12 Otherwise, targeted analytical methods are required in order to perform the isomer separation and their ratio determination, e.g., enantiomer determination of 13−15 D,L-SeMet. Efficient methods used for extensive Se speciation in yeast are described in the literature. They rely on multidimensional liquid chromatographic preconcentration of the metabolites with parallel detection by elementary (i.e., inductively coupled plasma mass spectrometry, ICP MS) and organic mass spectrometry (e.g., electrospray ionization MS, ESI MS). This strategy has been successfully used to determine the selenium speciation in Se-rich yeast. Various orthogonal chromatographic or electrophoretic separations have been used in order to access the most extensive Se-speciation possible8,9,16−27

elenium (Se) is an essential trace element involved in numerous metabolic functions, mainly related to oxidative stress.1 Nowadays, the Se-deficiency on diet may be counteracted by a supplementation based on “selenized” Saccharomyces cerevisiae yeast.2 Indeed, this yeast is able to convert a high amount of inexpensive inorganic Se in safe Se-containing organic compounds3 until there is 3000 μg of Se per gram of dried yeast.4 Several clinical trials (Nutritional Prevention of Cancer, NPC, and Prevention of Cancer by Intervention with SElenium, PREcise) suggested that Se supplementation with Se-rich yeast decreases the incidence of various kinds of cancer (e.g., lung and prostate cancer).5,6 Nonetheless, Se supplementation as L-selenomethionine (L-SeMet), with or without vitamin E, during the SELECT clinical trial (SElenium and vitamin E Cancer prevention Trial) did not prevent the prostate cancer occurrence,7 suggesting than L-SeMet was not the active form on Se-rich yeast during the two first clinical trials. Nowadays, more than 50 different Se-containing species have been identified in water Se-rich yeast extracts.8,9 Several Se-containing compounds identified in Se-rich yeast are also found as structural isomer mixtures mainly being related to oligopeptides and especially selenocystathionine derivatives (e.g., 2,3-dihydroxypropionylselenocystathionine, N-acetylselenocystathionine, γ-glutamylselenocystathionine). Interestingly, biological activity or absorption capabilities of Se-containing © 2014 American Chemical Society

Received: July 25, 2014 Accepted: October 8, 2014 Published: October 8, 2014 11246

dx.doi.org/10.1021/ac503142u | Anal. Chem. 2014, 86, 11246−11254

Analytical Chemistry

Article

Figure 1. Structures of some isomeric selenocompounds 2,3-dihydroxypropionylselenocystathionine extracted from Se-rich yeast. To the top left is the isomer “B” and isomer “A” is to the right. The 2 stable complexes with 4′-nitrobenzo-15-crown-5 ether are schematized to the bottom: B1 and B2 (left) and A1 and A2 (right). See the text for details and Figure S-2, Supporting Information, for the 3D pictures. Dashed lines are for electrostatic interactions. Stability energies of complexes are ranked as follows: A1 > A2 > B1 > B2.

increase the difference of CCS between isomers or (near) isobaric ions, based on the proof of concept of nonspecific arrival drift time shift proposed by Creaser and co-workers34 using amines and polyethers, Hilderbrand and co-workers,35 and Colgrave and co-workers.36 The objective of this paper is to describe the proof of concept of a new and original versatile analytical strategy where isomer ratio can be obtained from a complex mixture in a real sample (e.g., Se-rich yeast water extract). Ion mobility mass spectrometry and selective shift reagent were used to demonstrate the feasibility of reaching more advanced data in a speciation analysis context. A selective shift reagent is a ligand specifically modifying the CCS of ions by the formation of noncovalent complexes with only (ideally) target analytes. Multidimensional liquid chromatography was coupled to IMMS in order to separate the structural isomers of these Sespecies for isomer ratio determination. Multidimensional liquid chromatography allowed one to achieve preconcentration of the most abundant Se-species, presenting structural isomers and reducing the suppression of ionization during electrospray ionization. Coupling the last reverse phase LC dimension online with ion mobility mass spectrometry (IM-MS) was the key step for the separation of the structural isomers and the determination of their ratio. The most abundant isomer mixture described in the literature12,21 as 2,3-dihydroxypropionyl-selenocystathionine (2,3-DHP-selenocystathionine; m/ z: 359.0358; Figure 1) was selected as the model compound for

Isomer ratio determination is however challenging to achieve. An answer could be the use of a fast, orthogonal, and on line separation technique able to separate the isomers. Ion mobility is an additional orthogonal separation technique using the tridimensional structure of analytes to separate them as ions in the gas phase, according to their drift time. The separation into a buffer gas (or drift gas) requires only a few milliseconds. The drift time, in the hard spheres model, depends on the collisional cross section (CCS), which can be viewed as the averaged projection of the ion shape on a plane.28 Ion mobility cells coupled to mass spectrometry (IM-MS) and especially to time of flight instruments are commercially available and have low duty cycle and low detection limit. Kanu and co-workers29 and Lapthorn and co-workers30 reviewed the different types of instruments and their applications. Interestingly, the mobility coefficients of low molecular weight ions do not depend on the liquid phase composition because the solvent has little influence on their structure compared to the case of biopolymers31 having multiple charge states and tertiary/quaternary structures.32 For small ions, limited changes in conformation may lead to clear separations by IM-MS. In addition, Kune and co-workers has very recently introduced the concept of selective shift reagent (SSR) and ion mobility mass spectrometry as an isomer structural discrimination technique, functional-group probing, or structural elucidation.33 The separation in the mobility cell was improved by using host−guest molecular recognition to 11247



Article

Figure 2. Purification from a water Se-rich yeast extract (so-called batch “ES1149”) of 2,3-DHP-selenocystathionine by multidimensional LC (SEC/ SAX/RP) coupled with ICP MS or ESI MS; the fractions of interest are highlighted by a dotted rectangle. (A) Preconcentration of peptidic fraction containing selenometabolites fraction SEC ICP MS (Superdex75). (B) Preconcentration of 2,3-DHP-selenocystathionine by SAX-ICP MS (PRPX100). (C) Online RP HPLC-ESI-IM-MS for structural elucidation of 2,3-DHP-selenocystathionine. (D) RP HLPC-ESI mass spectrum at retention time = 3.27 min. 76

Se, 77Se, 78Se, (and 80Se if dynamic reaction cell and methane were used), 82Se, 83Kr, 88Sr, and 103Rh (See ICP MS settings in Table S-6, Supporting Information). The first chromatographic dimension was size exclusion chromatography (Superdex75 gel in a homemade glass column of 600 mm × 30 mm × 34 μm) in isocratic mode using 10 mM ammonium acetate buffer adjusted to pH 9.5, and flow rate was 1 mL·min−1. The second dimension was anion exchange chromatography (Hamilton PRP-X100 250 mm × 4 mm × 5 μm) using 10 and 250 mM ammonium acetate in gradient mode at pH 5.5 (see Table S-7, Supporting Information) using 1.5 mL·min−1 flow rate. The third LC dimension was capillary reverse phase liquid chromatography (Thermo-Fisher PepMapC18-100 column 150 mm × 0.3 mm × 5 μm) coupled to electrospray ion mobility mass spectrometry. Gradient mode was used with a flow rate of 3 μL·min−1 (see Table S-7, Supporting Information) and 2.34 mM formic acid and acetonitrile as mobile phase. Fractions were pooled, frozen, and freeze-dried between each sample preparation step. An 1100 series HPLC Agilent pump was used for all chromatographic separations. Electrospray MS detection was performed by coupling the HPLC system to a Waters Synapt-G2 HDMS (Waters Company, Manchester, U.K.) instrument operating in positive ionization (+3 kV) in the “resolution mode” (as reflectron in V mode). Mass calibration (m/z: 50−2000) was performed using phosphoric acid solution (0.1% H3PO4 mass/volume in a 50% acetonitrile solution). LM Quad was set to 20 during selection of m/z: 672.2 corresponding to 2,3-DHP-selenocystathionine

this purpose. Separation by ion-mobility of the isomers of 2,3DHP-selenocystathionine was attempted on the native compounds or after formation of a specific host−guest system (shifting reagent) to one of the isomers using crown ethers. The structures of selective complexes of shift reagent with 2,3DHP-selenocystathionine and their stability were calculated using computational chemistry methods.

■

EXPERIMENTAL SECTION Materials. All chromatographic eluents and reagents were purchased from Sigma-Aldrich (Bornem, Belgium) and were metal trace analysis grade except for acetonitrile (HPLC-MS grade). A Milli-Q ultrapure water system from Millipore (Millipore, Molsheim, France) was used throughout the study. Crown ethers (18-crown-6, benzo-18-crown-6, dibenzo-18crown-6, and 4′-nitrobenzo-15-crown-5) were purchased from VWR (Leuven, Belgium). The sample consisted in a batch of Se-rich yeast (Saccharomyces cerevisiae CNCM I-3060 batch ES1149) Sel-Plex (Alltech, Nicholasville, KY) corresponding to a yeast strain Saccharomyces cerevisiae grew on inorganic selenium. Extraction Procedure and Liquid Chromatography Purification of 2,3-DHP-selenocystathionine. Fully detailed experimental procedures for 2,3-dihydroxypropionylselenocystathionine purification are described in the Supporting Information and described elsewhere.37,38 In brief, isomers of 2,3-dihydroxypropionylselenocystathionine were obtained after a 3D LC fractionation using ICP MS detection and monitoring 11248



Article

Figure 3. Experimental design for the isomer ratio determination of 2,3-DHP-selenocystathionine using online HPLC−ESI-IM-MS. Postcolumn addition is performed using a syringe-pump and a Tee connector after chromatographic separation. Soft collision energy in the trap is used to dissociate the nonspecific host−guest complex and ion mobility to perform the gas phase separation where peak areas lead to the isomer ratio determination (Method #1). MS/MS after ion mobility separation is used in Method #2 for isomer ratio determination on the basis of the fragmentation pattern. See the text for details.

and 4′-nitrobenzo-15-crown-5 ether complexes [C10H19N2O7Se + C14H19NO7]+ as selective shift reagent. Table S-8, Supporting Information, provides the parameters used during ESI MS and ESI IM-MS experiments. Postcolumn addition of crown ether was done using a syringe pump (1 μL·min−1) via a PEEK Tee connector and PEEK tubing (70 cm × 100 μm I.D.). Crown ether was dissolved in acetonitrile to a final concentration of 1 mM. The 10 mM stock solutions were prepared in dimethyl sulfoxide. Ion Mobility Separation. Ion mobility separation was tested with and without crown ethers as selective shift reagents. When using crown ethers, a postcolumn addition (1 mM of crown ether was added to the mobile phase with a Tee connector and syringe pump after chromatographic separation; see Figure 3) was used. On the basis of this strategy, 2 methods were developed. Method #1 involves the dissociation of the unstable crown ether complex by setting a 4 V collision energy in the trap cell prior to ion mobility separation. Ion mobility parameters are given in Table S-8, Supporting Information. On the basis of Method #1, a second method (i.e., Method #2) of separation/quantification was developed using fragmentation in the transfer cell. The experimental setup involved the selection of the expected complex between 2,3-DHP-selenocystathionine and 4′-nitrobenzo-15-crown-5 ether (m/z: 672.2) by the Synapt G2 quadrupole, the dissociation of the unstable complex by a 4 V collision energy followed by ion mobility separation. Finally, the transfer cell was used to fragment the isomers by

applying an energy ramp of 30 to 60 V. The different analytical strategies are depicted in Figure 3. Software and Theoretical Computing. Geometry optimization and frequency calculations were obtained from density functional theory (Gaussian09) using B3LYP/6-31G +(d,p) functional and basis set. Absolute energies in Hartree (Ha): molar heat capacity (Cv), enthalpy (H), free Gibbs energy (G), and entropy (S) were computed using Gaussian, i.e., at ground state (absolute zero temperature) and then at 298 K. ΔH, ΔG, and ΔS at higher temperature were then calculated (See Tables S-2, S-3, and S-4, Supporting Information, for details). Steric energies were obtained from MM2 molecular mechanics. Peak deconvolutions were performed using PeakFit v4.11.

■

RESULTS AND DISCUSSION Extraction Procedure and 3 Dimensional Liquid Chromatography Purification of 2,3-DHP-selenocystathionine. Once extracted by water, 2,3-DHP-selenocystathionine was first purified by size exclusion chromatography (SEC). The obtained chromatogram on Superdex75 gel (see Figure2A) approximately exhibits the same separation pattern of Secompounds than in the literature on Superdex 30 gel39 with the presence of protein fractions and low molecular weight fractions. In the present work, the chromatographic separation efficiency was better in the protein fraction than the metabolic fraction. For this reason, the selected chromatographic peak containing 2,3-DHP-selenocytathionine (framed in the chro11249



Article

Figure 4. Arrival drift time distribution of 2,3-DHP-selenocystathionine (m/z: 359.04) during the RP-HPLC−ESI-IM-MS experiment. (A) Arrival time of m/z: 359.04 without the use of crown ether addition. (B) Arrival time of m/z: 359.04 with 4′-nitrobenzo-15-crown-5 ether postcolumn addition. Peak areas at dt = 6.07 ms (drift time) and dt =11.18 ms were 87% and 13%, respectively. (C) Mass spectrum at dt = 6.07 ms. (D) MS/MS spectrum at dt = 6.07 ms. (E) Mass spectrum at dt = 11.18 ms. (F) MS/MS spectrum at dt = 11.18 ms. Dotted rectangles show some of the characteristic MS/MS fragments from isomers of 2,3-DHP-selenocystathionine.

removal of most of the ammonium ions. Indeed, ammonium has a high binding capability with crown ethers and could prevent the binding of the selective shift reagent with the selenometabolites. Ion Mobility Separation of 2,3-dihydroxypropionylselenocystathionine Isomers. Ion mobility separation of 2,3-DHP-selenocystathionine isomers was tested according to 2 different methods: (1) isomers separation by ion-mobility on the native (i.e., free) compounds and (2) isomers separation by ion-mobility after formation of a specific complex to one of the isomers using crown ethers. A flow chart of the experimental method for the isomer ratio determination is provided in Figure 3. In the first approach, the separation of the coeluting isomers of 2,3-DHP-selenocystathionine was tested on the native isomers (i.e., free species). The drift time distribution is presented in Figure 4A. The mobility peak centered at 1.86 ms, corresponding to both of the isomers of 2,3-DHP-selenocystathionine, showed very poor separation on the isomers (see Figure 4A). Indeed, the limited ion mobility resolution (RCCS/ΔCCS ≈ 40−50 according to the IM-MS specification) did not allow the separation of 2,3-DHP-selenocystathionine isomers due to the small difference of collision cross sections between both isomers (110 and 108 Å2, i.e., roughly 2% of the CCS difference or RCCS/ΔCCS > 60). The CCS theoretical values were obtained after structure optimization by MM2 in Chem

matogram in Figure 2A) also contained molecules from glutathione metabolism and Se-oligopeptides.8,21 The low molecular weight fraction purified by SEC was then further purified by strong anion exchange chromatography (SAX). SAX was chosen, in agreement with the literature,40 as the second orthogonal chromatographic separation for its good separation performance on this low molecular weight fraction.8 As expected, the obtained chromatogram (see Figure 2B) revealed an intense peak having a retention time at 12 min assumed to be 2,3-DHP-selenocystathionine (framed in the chromatogram). Finally, the 2D-LC purified fraction underwent a last chromatographic step consisting of a reverse phase chromatography (RP). RP was chosen as the third and final chromatographic step in order to remove salts originating from the SAX chromatography and to allow an additional orthogonal chromatographic separation to avoid as much as possible the ion suppression affecting ESI sources. The chromatogram and mass spectrum showed the presence of 2,3-DHP-selenocystathionine (see Figure 2C,D), as verified by the presence of both the isotopic pattern and the mass defect characteristic to selenium in the mass spectrum related to the chromatographic peak. The identity of the species was also confirmed by its exact mass (δmass = 22 ppm in Figure 2D and 14 ppm in Figure 4C) and the presence of fragments in very good agreement with the literature.21,40 The last reverse phase LC dimension, using acetonitrile and formic acid as mobile phases, allows for the 11250



Article

tally was the isomer B and 4′-nitrobenzo-15-crown-5 ether host−guest system. This result is in full agreement with the theoretical energies obtained by computational chemistry. Moreover, the calculated ΔE between the 2 host−guest systems was roughly 30 kJ mol−1 where the isomer B and crown ether complex was less stable (see Table S-3, Supporting Information, for details). Ion Mobility Separation with Crown Ether: Method #2. The second method developed for the determination of the isomer composition of 2,3-DHP-selenocystathionine was based on their fragmentation patterns. Indeed, this method was identical to Method #1 (i.e., postcolumn addition of 4′nitrobenzo-15-crown-5 ether at 1 mM, collision induced dissociation of the less stable complex using soft collision energy into the trap, and then ion mobility separation) except that both isomers were fragmented after separation in the ion mobility cell using a ramping collision energy (30 to 60 V) in the transfer cell. As a result, a fragmentation pattern (based on a list of fragments described in the literature concerning 2,3DHP-selenocystathionine21,40) could be determined for both isomers at their respective drift times (see Figure 4D,F). The intensities of all described fragments (e.g., m/z: 56.0495, 88.0393, 135.9660, 137.9816, 176.0553, 181.9715, 183.9871, 269.9876, and 271.0192) were followed and reported on histograms (see Figure 5). Finally, the intensities of both isomers were added to set up a so-called “averaged fragmentation pattern” which corresponds to the fragmentation pattern that would be obtained without ion mobility separation. The obtained patterns showed significant differences between both isomers. As both forms contribute to the relative intensity of each fragment in the averaged profile, the isomer ratio could be determined by comparing the fragmentation patterns at specific drift times versus the averaged one (see Figure 5). The formula used to calculate the ratio was inspired from the isotope dilution quantification formulas.42 This method actually builds some systems of linear equations with 2 unknowns as below:

3D pro and Gaussian09 B3LYP/6-31G+(d,p), (see Table S-1, Supporting Information, for details). In order to improve the IM separation, a strategy based on the formation of a specific host−guest system of one of the isomers was set up by using crown ether as a specific shift reagent. By forming this specific host−guest complex, the isomer attached to the crown ether would appear at an apparent higher collision cross section than the free isomer. The new difference of CCS leads to fully resolved peaks in IMS (see Table S-1, Supporting Information). Considering that crown ethers are able to bind to primary amino groups,41 one could assume that the position of the chemical groups within the isomer (compared to the Se position) would affect the ability of the crown ether to bind 2,3-DHP-selenocystathionine with higher or lower affinity because of, but not exclusively, steric hindrance. To this end, 4 different crown ethers (18crown-6, benzo-18-crown-6, dibenzo-18-crown-6, and 4′-nitrobenzo-15-crown-5) were experimentally tested on Met-ArgPhe-Ala oligopeptide (MRFA). The choice of MRFA as the molecule model was driven by the presence of steric hindrance near/close to the complexing site, roughly mimicking the expected complexation behavior of 2,3-DHP-selenocysthationine. Concerning the crown ethers, we tested different steric hindrance effects and cycle size. 4′-nitrobenzo-15-crown-5 ether was also tested to explore the effect of the nitro group on the stability of the formed complex using molecular mechanics simulation (MM2) and Density Function Theory. These theoretical data lead to the selection of nitrobenzo-15-crown5 ether as SSR among the other tested crown ethers. The theoretical stability (in terms of “absolute energy”) and ΔE of each host−guest system between 4′-nitrobenzo-15-crown-5 ether and 2,3-DHP-selenocystathionine isomers were obtained using DFT and are presented in Table S-3, Supporting Information. From an experimental point of view, the selective shift reagent 4′-nitrobenzo-15-crown-5 ether (1 mM in 100% acetonitrile) was brought into the system by a postcolumn addition after the chromatographic separation. Coupling reverse phase LC as the last dimension with IM-MS was mandatory to avoid crown ether consumption by residual cationic salts (i.e., sodium, potassium, and ammonium) from the sample prior to complex formation with the target analytes. Ion Mobility Separation with Crown Ether: Method #1. The first method to determine the isomer composition of 2,3-DHP-selenocysthationine consisted of a postcolumn addition of 4′-nitrobenzo-15-crown-5 ether (1 mM), followed after the ionization by a soft collision induced dissociation of the less stable complex in the trap cell (4 V) and finally ion mobility separation as illustrated in Figure 3. A collision energy set to 4 V on the trap cell does not lead to any fragmentation of 2,3-DHP-selenocystathionine precursor ion. The obtained isomer ratio determination and the arrival drift time distribution are presented in Figure 4. In this case, the mobility data show a clear separation between both isomers with one mobility peak centered at 6.07 ms and the other centered at 11.18 ms (see Figure 4B). The identities of the free and complexed forms were verified by the acquisition of MS/MS spectra at respective drift times (see Figure 4D−F). The presence of fragments considered as characteristic21,40 allowed one to identify the free and complexed structures. Finally, the integration of the mobility peaks led to the determination of the isomer ratio (13% for isomer A and 87% for isomer B; see Figure 4B) assuming that the ionization efficiency was similar for both isomers. The unstable complex observed experimen-

[isomer A] × α%used fragment + [isomer B] × β %used fragment = ω%used fragment

where α% represents the relative intensity of the considered fragment in the recombined MS/MS spectrum due to isomer A, β% represents the relative intensity of the same fragment in the recombined MS/MS spectrum coming from isomer B, and ω% represents the relative intensity of this fragment in the recombined spectrum due to both isomers. Roughly each fragment could be used to calculate the isomer ratio. The values were averaged on all fragments to determine the isomer ratio. The results obtained by this second method (11% isomer B; 89% isomer A; see Figure S-3, Supporting Information) are in good agreement with those obtained by the first approach, i.e., without MS/MS after IMS (13% isomer B; 87% isomer A). Note that resolving these equation systems using a matrices calculation leads to a similar result (data not shown). Method Validation. The lack of commercially available authentic standards does not allow the method validation. The synthesis of such Se-compounds also seems problematic because of nontrivial reaction pathways in order to obtain the expected organoselenium compounds.43 Nevertheless, Dernovics and co-workers,40 after several chromatographic purification steps followed by parallel ICP MS and ESI-MS detection, succeeded to slightly separate the isomers of 2,311251



Article

isomer A and B, respectively, which fits with the results presented here (see Figure S-4, Supporting Information). Computational Chemistry Results. The structures of isomers A and B alone were first optimized using DFT or MM2. Then, the addition of the nitrobenzo-15-crown-5 ether as SSR during the calculation was performed to evaluate the ability of the SSR to specifically bind one of the isomers of 2,3DHP-selenocystathionine. The general methodology to obtain all stable structures is to use molecular mechanics and molecular dynamics by MM2 before structure reoptimization and energy calculations (ΔH, ΔG, ΔS, and Cv) using DFT. MM2 simulations were performed to evaluate the accuracy of the modeling when the use of low computing time is required. A relatively small system with MM2 modeling is less timeconsuming compared to DFT. MM2 simulations suggested that the primary amino group of 2,3-DHP-selenocystathionine could strongly interact with the crown ethers as expected. Furthermore, the 2,3-dihydroxypropionyl moiety was found to also interact with the crown ethers (see Figure 1 and Figures S-1 and S-2, Supporting Information). The MM2 simulation also strongly suggested a possible interaction of the nitrobenzo moiety of the SSR with the selenometabolite of interest. Calculations performed by DFT were roughly consistent with the results obtained using MM2 modeling. The 4′-nitrobenzo-15-crown-5 ether was found to provide 2 different stable ligand/receptor systems with each of the 2,3-DHP-selenocystathionine isomers (isomers A and B), i.e., four different complexes denoted as complexes A1 or A2 and B1 or B2 (see Figure 1 and Figures S-1 and S-2, Supporting Information). One is the interaction between the primary amino group of one of the 2,3-DHP-selenocystathionine and the poly(ethylene oxide) ring moiety of the crown ether (i.e., complexes A1 or B1). The other is the interaction with the alcohol functional group (from 2,3-dihydroxypropionyl moiety) and the crown ether’s ring (i.e., isomers A2 and B2). The energies determination by DFT suggests that the complexes A1 and B1 (i.e., primary amino group from isomer A or B, respectively, interacting with the poly(ethylene oxide) ring) are more stable than A2 and B2, respectively. Moreover, the complex A1 (i.e., primary amino group from isomer A with polyethylene ring of 4′-nitrobenzo-15-crown-5 ether) was found to be more stable than complex B1 (ΔGDFT ≈ 30−50 or 70 kJ mol−1 according to the considered chemical reaction, see Tables S-3 and S-4 for details, Supporting Information). In short, the stability energies for the complexes were A1 > A2 > B1 > B2. Interestingly, MM2 made pretty accurate predictions in terms of the location of the interaction between the crown ethers and the 2,3-DHP-selenocystathionine. The calculated CCS from the structures obtained using MM2 were very close to the CCS obtained after structure reoptimization using DFT calculations (less than 2% of CCS difference, data not shown). The estimated energies of the systems after MM2 modeling (ΔSteric EnergyMM2 ≈ 5 kJ mol−1 in favor of isomer A complex formation; see Table S-5, Supporting Information) were in agreement with the values obtained using DFT. The difference of energy obtained using MM2 was indeed of the same sign as the energy difference obtained after the DFT calculation. Steric energy determined by MM2 was roughly 10−20% of the total energy (as ΔG) calculated using DFT. The energy values obtained after DFT calculations were used in order to estimate the relative stability (in terms of energies’ delta) of all kinds of interaction between 2,3-DHP-

Figure 5. Isomer ratio determination on the basis of Method #2 (see the text for details). Absolute intensities of fragments from 2,3-DHPselenocystathionine were reported for isomer A (A) and isomer B (B). The averaged fragmentation pattern of isomer A and isomer B was determined (C). Linear systems of equations with 2 unknowns were built to access the isomer ratio determination (see the text for details).

DHP-selenocystathionine by hydrophilic interaction liquid chromatography (HILIC) while working on the purification of several Se-metabolites. The Saccharomyces cerevisiae CNCM I-3060 batch ES453 of Se-rich yeast used by Dernovics and coworkers was produced by the same manufacturer and was described to be closely related (in terms of selenocompound composition) to the ES1149 batch used for this study.8,44 The value extracted from the deconvolution of raw data (available at the laboratory) allowed one to determine a ratio of 5%:95% for 11252



Article

cross sections of the isomers. However, the use of a selective shift reagent (4′-nitrobenzo-15-crown-5 ether) as specific host−guest reagent to a particular isomer allowed one to perform the separation and quantification of the isomer ratio using the integration of the mobility peaks or the so-called “fragmentation patterns” method. Both methods provide results which are in good agreement to one another (13−87% and 11−89%, respectively). In the case of 2,3-DHP-selenocystathionine, the major isomer was isomer B (see Figure 4). The values presented in this study are in good agreement with (1) theoretical values obtained by computational chemistry and (2) the isomer ratio determined from raw data by Dernovics and co-workers after slight separation of this selenocompound extracted from a similar batch of Se-rich yeast by HILIC chromatography coupled to high resolution mass spectrometry.40 The addition of selective shift reagent opens up new potential applications of ion mobility in the field of separation of isobaric compounds, which are characterized by unresolved collisional cross section differences without coordination complex formation. Moreover, this work demonstrated that a higher level of sample characterization could be obtained (i.e., isomer ratio determination) during speciation analysis of a trace element by the use of ion mobility mass spectrometry and the use of selective shift reagents, i.e., the determination of isomer ratio in a single analysis. Ion mobility separation avoids the addition of a tedious development of a new devoted orthogonal separation for the isomer ratio determination. Indeed, the separation of structural isomers is often considered as a very challenging analytical issue because of the requirement of a dedicated separative method. Furthermore, the separation of ions sharing a similar collision cross section by IMS can be directly implemented for the determination of isomer composition only by optimizing the nature of the mobilityshift reagent: steric hindrance, (un)favorable electrostatic interactions, Van der Walls interactions, and so on. This optimization can be performed either empirically or theoretically, using computational chemistry modeling methods. Method validation is still lacking because 2,3-dihydroxypropionylselenocystathionine is currently not commercially available. Nonetheless, recent advances in selenometabolites synthesis lead to the synthesis of 2,3-dihydroxypropionylcontaining selenocysteine or glutathione45 that should help to confirm the results of the proposed methodology in the future.

selenocystathionine and 4′-nitrobenzo-15-crown-5 ether (i.e., complexes A1, A2, B1, and B2; see Figure 1). These values are provided in Tables S-3 and S-4, Supporting Information. The theoretical data were in good agreement with the experimental results obtained during this work. Indeed, the most stable complexes obtained after ion mobility separation in the gas phase were consistent with the theoretical values obtained by computational chemistry. One should note that the resolution of the traveling wave IMS used during this work should fully differentiate the complexes B1 from B2 or complexes A1 from A2, respectively, but neither A1 from B1 nor A2 from B2 (see Tables S-1 and S-4, Supporting Information). No additional peaks were observed during the IMS separation of the analytes (see Figure 4), suggesting than the collision energy applied on the trap cell (see Figure 3) was efficient enough to remove the unstable complexes. Actually, any kind of mixture containing free isomer A and complex B1 or B2 (less stable than A1 or A2) will lead to the formation of largely more stable complex A1 or A2 after activation in the trap. The results of theoretical chemistry (see Table S-4, Supporting Information) pointed out the favorable complex formation of 4′-nitrobenzo-15-crown-5 ether and the isomer A, as observed in our experimental data (see Figure S-5, Supporting Information). At this stage of investigation, the small difference of CCS obtained for the most stable host−guest systems, i.e., complexes A1 and A2 (see Table S-1, Supporting Information), should lead to unresolved peaks after ion mobility separation, but both contributed to the quantification of the same isomer of 2,3-DHP-selenocystathionine. Into the trap cell, the less stable host−guest systems, i.e., complexes B1 and B2, should work as crown ether donors to isomer A during the isomer ratio determination and do not induce non-negligible analytical bias for the isomer ratio determination. The most abundant isomer of 2,3-DHP-selenocystathionine experimentally determined was the isomer B (see Figure 1). Interestingly, the theoretical value of energy delta to form the free isomer A or isomer B is roughly 40 kJ mol−1 (see Table S2, Supporting Information, for details) in favor of isomer B formation, in good agreement with the experimental results. Considering the experimental values compared to the theoretical data, computational chemistry tools could be useful in order to predict and/or design the SSR structure before isomer separation attempts using ion mobility spectrometry. The relative amount of 2,3-DHP-selenocystathionine isomer determined experimentally (≈90% and 10% for isomer A and B, respectively) is also in good agreement with the thermodynamically favorable formation of the isomer B determined by computational chemistry.

■

ASSOCIATED CONTENT

S Supporting Information *

■

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

■

CONCLUSIONS The analytical methods developed during this work allowed one to determine the isomer composition of the major Semetabolite (presenting a structural isomerism) extracted from Se-rich yeast, namely, the 2,3-dihydroxypropionylselenocystathionine. Those methods relied on a tridimensional liquid chromatographic purification of the metabolite of interest followed by gas phase ion mobility separation to perform the isomer separation. The LC purification steps (SEC-SAX-RP) were based on the state-of-the-art of Se-speciation in Se-rich yeast and adapted to obtain a high amount of fairly pure 2,3DHP-selenocystathionine. Traveling wave ion mobility did not successfully separate the native isomers of 2,3-DHP-selenocystathionine, most likely because of a small difference in collision

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Rayman, M. P. Lancet 2000, 356, 233−241. (2) Dumont, E.; Vanhaecke, F.; Cornelis, R. Anal. Bioanal. Chem. 2006, 385, 1304−1323. (3) Schrauzer, G. N. Pure Appl. Chem. 2006, 78, 105−109. (4) Ouerdane, L.; Mester, Z. J. Agric. Food. Chem. 2008, 56, 11792− 11799. 11253



Article

(5) Clark, L. C.; Combs, G. F., Jr.; Turnbull, B. W.; Slate, E. H.; Chalker, D. K.; Chow, J.; Davis, L. S.; Glover, R. A.; Graham, G. F.; Gross, E. G.; Krongrad, A.; Lesher, J. L., Jr.; Park, H. K.; Sanders, B. B., Jr.; Smith, C. L.; Taylor, J. R. JAMA 1996, 276, 1957−1963. (6) Clark, L. C.; Dalkin, B.; Krongrad, A.; Combs, G. F., Jr.; Turnbull, B. W.; Slate, E. H.; Witherington, R.; Herlong, J. H.; Janosko, E.; Carpenter, D.; Borosso, C.; Falk, S.; Rounder, J. Br. J. Urol. 1998, 81, 730−734. (7) Lippman, S. M.; Klein, E. A.; Goodman, P. J.; Lucia, M. S.; Thompson, I. M.; Ford, L. G.; Parnes, H. L.; Minasian, L. M.; Gaziano, J. M.; Hartline, J. A.; Parsons, J. K.; Bearden, J. D., III; Crawford, E. D.; Goodman, G. E.; Claudio, J.; Winquist, E.; Cook, E. D.; Karp, D. D.; Walther, P.; Lieber, M. M.; Kristal, A. R.; Darke, A. K.; Arnold, K. B.; Ganz, P. A.; Santella, R. M.; Albanes, D.; Taylor, P. R.; Probstfield, J. L.; Jagpal, T. J.; Crowley, J. J.; Meyskens, F. L., Jr.; Baker, L. H.; Coltman, C. A., Jr. JAMA, J. Am. Med. Assoc. 2009, 301, 39−51. (8) Casal, S. G.; Far, J.; Bierla, K.; Ouerdane, L.; Szpunar, J. Metallomics 2010, 2, 535−548. (9) Preud’Homme, H.; Far, J.; Gil-Casal, S.; Lobinski, R. Metallomics 2012, 4, 422−432. (10) Sohn, O. S.; Fiala, E. S.; Upadhyaya, P.; Chae, Y. H.; ElBayoumy, K. Carcinogenesis 1999, 20, 615−621. (11) Sohn, O. S.; Li, H.; Surace, A.; El-Bayoumy, K.; Upadhyaya, P.; Fiala, E. S. Anticancer Res. 1995, 15, 1849−1856. (12) Dernovics, M.; Far, J.; Lobinski, R. Metallomics 2009, 1, 317− 329. (13) Day, J. A.; Kannamkumarath, S. S.; Yanes, E. G.; Montes-Bayón, M.; Caruso, J. A. J. Anal. At. Spectrom. 2002, 17, 27−31. (14) Devos, C.; Sandra, K.; Sandra, P. J. Pharm. Biomed. Anal. 2002, 27, 507−514. (15) Ilisz, I.; Berkecz, R.; Péter, A. J. Sep. Sci. 2006, 29, 1305−1321. (16) Casiot, C.; Vacchina, V.; Chassaigne, H.; Szpunar, J.; PotinGautier, M.; Łobiński. Anal. Commun. 1999, 36, 77−80. (17) Chassaigne, H.; Chery, C. C.; Bordin, G.; Vanhaecke, F.; Rodriguez, A. R. J. Anal. At. Spectrom. 2004, 19, 85−95. (18) Dernovics, M.; Lobinski, R. J. Anal. At. Spectrom. 2007, 23, 72− 83. (19) Dernovics, M.; Lobinski, R. Anal. Chem. 2008, 80, 3975−3984. (20) Dernovics, M.; Ouerdane, L.; Tastet, L.; Giusti, P.; Preud’Homme, H.; Lobinski, R. J. Anal. At. Spectrom. 2006, 21, 703−707. (21) Far, J.; Preud’homme, H.; Lobinski, R. Anal. Chim. Acta 2010, 657, 175−190. (22) Infante, H. G.; O’Connor, G.; Rayman, M.; Hearn, R.; Cook, K. J. Anal. At. Spectrom. 2006, 21, 1256−1263. (23) Infante, H. G.; O’Connor, G.; Rayman, M.; Wahlen, R.; Entwisle, J.; Norris, P.; Hearn, R.; Catterick, T. J. Anal. At. Spectrom. 2004, 19, 1529−1538. (24) Infante, H. G.; O’Connor, G.; Rayman, M.; Wahlen, R.; Spallholz, J. E.; Hearn, R.; Catterick, T. J. Anal. At. Spectrom. 2005, 20, 864−870. (25) Kotrebai, M.; Birringer, M.; Tyson, J. F.; Block, E.; Uden, P. C. Anal. Commun. 1999, 36, 249−252. (26) Mounicou, S.; McSheehy, S.; Szpunar, J.; Potin-Gautier, M.; Lobinski, R. J. Anal. At. Spectrom. 2002, 17, 15−20. (27) Palacios, Ò .; Ruiz Encinar, J.; Schaumlöffel, D.; Lobinski, R. Anal. Bioanal. Chem. 2006, 384, 1276−1283. (28) Howdle, M. D.; Eckers, C.; Laures, A. M. F.; Creaser, C. S. Int. J. Mass Spectrom. 2010, 298, 72−77. (29) Kanu, A. B.; Dwivedi, P.; Tam, M.; Matz, L.; Hill, H. H., Jr. J. Mass Spectrom. 2008, 43, 1−22. (30) Lapthorn, C.; Pullen, F.; Chowdhry, B. Z. Mass Spectrom. Rev. 2013, 32, 43−71. (31) Chen, Y. H.; Hill, H. H.; Wittmer, D. P. J. Microcolumn Sep. 1994, 6, 515−524. (32) Li, J.; Taraszka, J. A.; Counterman, A. E.; Clemmer, D. E. Int. J. Mass Spectrom. 1999, 185−187, 37−47. (33) Kune, C.; Far, J.; Delvaux, C.; Eppe, E.; De Pauw, E. Contribution of ion mobility for structural analysis and analytical

chemistry: Use of selective IMS shift reagents (SSR). Poster session presented at 62nd ASMS Conference on Mass Spectrometry and Allied Topics, Baltimore, USA (Maryland), June 15−19, 2014. (34) Creaser, C. S.; Griffiths, J. R.; Stockton, B. M. Eur. J. Mass Spectrom. 2000, 6, 213−218. (35) Hilderbrand, A. E.; Myung, S.; Clemmer, D. E. Anal. Chem. 2006, 78, 6792−6800. (36) Colgrave, M. L.; Bramwell, C. J.; Creaser, C. S. Int. J. Mass Spectrom. 2003, 229, 209−216. (37) Encinar, J. R.; Sliwka-Kaszynska, M.; Polatajko, A.; Vacchina, V.; Szpunar, J. Anal. Chim. Acta 2003, 500, 171−183. (38) García-Reyes, J. F.; Dernovics, M.; Ortega-Barrales, P.; Fernández-Alba, A. R.; Molina-Díaz, A. J. Anal. At. Spectrom. 2007, 22, 947−959. (39) Encinar, J. R.; Ouerdane, L.; Buchmann, W.; Tortajada, J.; Lobinski, R.; Szpunar, J. Anal. Chem. 2003, 75, 3765−3774. (40) Dernovics, M.; Far, J.; Lobinski, R. Metallomics 2009, 1, 317− 329. (41) Chen, Y.; Rodgers, M. T. J. Am. Chem. Soc. 2012, 134, 2313− 2324. (42) Rodríguez-González, P.; Marchante-Gayón, J. M.; García Alonso, J. I.; Sanz-Medel, A. Spectrochim. Acta, Part B: At. Spectrosc. 2005, 60, 151−207. (43) Campbell, T. W.; Walker, H. G.; Coppinger, G. M. Chem. Rev. 1952, 50, 279−349. (44) Far, J.; Bérail, S.; Preud’Homme, H.; Lobinski, R. J. Anal. At. Spectrom. 2010, 25, 1695−1703. (45) Egressy-Molnar, O.; Magyar, A.; Gyepes, A.; Dernovics, M. RSC Adv. 2014, 4, 27532−27540.

11254


The Use of Ion Mobility Mass Spectrometry for Isomer Composition

Recommend Documents