ac research
Anal. Chem. 2008, 80, 3975–3984
Accelerated Articles Speciation Analysis of Selenium Metabolites in Yeast-Based Food Supplements by ICPMSsAssisted Hydrophilic Interaction HPLCsHybrid Linear Ion Trap/Orbitrap MSn Mihaly Dernovics† and Ryszard Lobinski*,‡,§ Department of Applied Chemistry, Corvinus University of Budapest, 1118 Budapest Villa´nyi u´t 29-33, Hungary, Laboratoire de Chimie Analytique Bio-inorganique et Environnement, CNRS UMR 5254, He´lioparc, 2, Avenue Pr. Angot, F-64053 Pau, France, and Department of Analytical Chemistry, Warsaw University of Technology, 00-664 Warszawa, Poland A new method was proposed for speciation analysis for selenium metabolites in Se-rich yeast. The coupling of a normal bore (4.6 mm) hydrophilic interaction liquid chromatography (HILIC) column with a hybrid linear ion trap/orbital ion trap mass spectrometer allowed the detection of the selenium-isotopic pattern in mass spectra down to the intrascan abundance of 0.001 with the lowand sub-part per million (ppm) mass accuracy regardless of the concentration. The quantitative elution recovery was verified by online ICPMS. The confirmation that all the species present were found was achieved by the parallel use of anion-exchange HPLC-ICPMS optimized for the maximum resolution. The species with intrascan abundance of at least 0.005 produced a cascade of product ion mass spectra to at least MS4 with the preservation of the selenium isotopic pattern and the sub-ppm mass accuracy, which largely facilitated the structure elucidation. The approach was successfully applied to the characterization of nine (all which were present in the analyzed sample) selenium species in one chromatographic run. The essential role of selenium, its implication in cancer prevention, and the low levels of this element in the diet in many countries are at the origin of the increasing interest in the supplementation of food * Corresponding author. E-mail:
[email protected]. Phone: +33 559407755. Fax: +33 559-407781. † Corvinus University of Budapest. ‡ CNRS UMR 5254. § Warsaw University of Technology. 10.1021/ac8002038 CCC: $40.75 2008 American Chemical Society Published on Web 04/26/2008
and feed with this element.1,2 The principal source of “organic” selenium is yeast grown in the presence of selenite.3 Whereas most of selenium is expected to be incorporated by proteins, its significant fraction in the marketed supplements is found as a mixture of low molecular weight metabolites.4 The knowledge of virtually all forms present at levels exceeding 0.1% is required to comply with regulations of governmental agencies issuing sales authorizations.5 The set of selenium metabolites is also a precious tracer of the origin of the product and an important parameter of the biotechnological process.6 Therefore, there is a considerable interest in the development of methods allowing the qualitative and quantitative characterization of the “selenometabolome” in yeast and other biological samples, which is in line with the overall trend of exhaustive profiling of all metabolitespresent in a target organism, referred to as metabolomics.7,8 Mass spectrometric methods, usually preceded by HPLC, offer unique advantages for the identification and quantification of metabolites. Metabolite structures can be elucidated on the basis of the empirical formulas of the parent compound and fragment ions (data provided by high-resolution and high-accuracy electrospray MS) and of the lineage of fragment ions observed in (1) (2) (3) (4) (5) (6) (7) (8)
Combs, G. F. Br. J. Nutr. 2001, 85, 517–547. Rayman, M. P. Lancet 2000, 356, 233–241. Schrauzer, G. N. J. Am. Coll. Nutr. 2001, 20, 1–4. Larsen, E. H.; Hansen, M.; Paulin, H.; Moesgaard, S.; Reid, M.; Rayman, M. J. AOAC Int. 2004, 87, 225–232. Eur. Food Saf. Authority J. 2006, 430, 1–23. Schrauzer, G. N. Pure Appl. Chem. 2006, 78, 105–109. Szpunar, J. Analyst 2005, 130, 442–465. Fan, T. W. M.; Higashi, R. M.; Lane, A. N. Drug Metab. Rev. 2006, 38, 707–732.
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tandem MS and especially MSn.9 On the other hand, quantification in electrospray MS is not straightforward and requires the availability of standards or a metabolite-tagging strategy. Hence the growing popularity of ICPMS, which provided that a to-bedetermined metabolite contains a heteroatom (S, P, metal, or metalloid), allows the quantitative determination of the metabolite without knowing its identity.10 Regarding identification, the reliable determination of the empiric formula requires less than 2 ppm accuracy on a routine basis. Such levels of mass accuracy were demonstrated to be achievable by time-of-flight (TOF) instruments only for pure analytes in ideal conditions when mass-lock calibration was used.11,12 In practice, for the analysis of biological extracts, mass accuracies were reported between 10-20 ppm11,13 and values higher than 50 ppm were not rare, especially in the case of minor analytes.14,15 This is because of the particular susceptibility of TOF analyzers to variations in ion intensity due the fast acquisition systems with inherently modest dynamic range.16–18 In TOF MS, the 5 ppm mass accuracy is unlikely to be achieved over a signal intensity range larger than a few hundred in 1 s acquisitions in the ion counting mode, even when advanced algorithms for intensity correction were employed.19 The insufficiency of TOF accuracy for compound identification has been compensated by lineage of ions in the tandem MS (Q-TOF) mode. A number of reports showed successful applications of electrospray Q-TOF MS for the identification of some metabolites of arsenic in marine biota,20–22 of selenium in yeast14,15,23–25 and plants,26–29 and of metal complexes in plants and human sera.30,31 Usually, one or a few compounds out of a dozen present could only be identified. Multidimensional chromatography was required to achieve a sufficient purity of the analyte to obtain sufficient signal intensity. (9) Feng, X.; Siegel, M. M. Anal. Bioanal. Chem. 2007, 389, 1341–1363. (10) Lobinski, R.; Schaumloffel, D.; Szpunar, J. Mass Spectrom. Rev. 2006, 25, 255–289. (11) Satomi, Y.; Kudo, Y.; Sasaki, K.; Hase, T.; Takao, T. Rapid Commun. Mass Spectrom. 2005, 19, 540–546. (12) Ko ¨feler, H. C.; Gross, M. L. J. Am. Soc. Mass Spectrom. 2005, 16, 406–408. (13) Canas, I. R.; Hamilton, B.; Amandi, M. F.; Furey, A.; James, K. J. J. Chromatogr., A 2004, 1056, 253–256. (14) Lindemann, T.; Hintelmann, H. Anal. Chem. 2002, 74, 4602–4610. (15) McSheehy, S.; Szpunar, J.; Haldys, V.; Tortajada, J. J. Anal. At. Spectrom. 2002, 17, 507–514. (16) Blom, K. F. Anal. Chem. 2001, 73, 715–719. (17) Wu, J. J.; McAllister, H. J. Mass Spectrom. 2003, 38, 1043–1053. (18) Colombo, M.; Sirtori, F. R.; Rizzo, V. Rapid Commun. Mass Spectrom. 2004, 18, 511–517. (19) Makarov, A.; Denisov, E.; Lange, O.; Horning, S. J. Am. Soc. Mass Spectrom. 2006, 17, 977–982. (20) McSheehy, S.; Szpunar, J.; Lobinski, R.; Haldys, V.; Tortajada, J.; Edmonds, J. S. Anal. Chem. 2002, 74, 2370–2378. (21) Pickford, R.; Miguens-Rodriguez, M.; Afzaal, S.; Speir, P.; Pergantis, S. A.; Thomas-Oates, J. E. J. Anal. At. Spectrom. 2002, 17, 173–176. (22) Pergantis, S. A.; Wangkarn, S.; Francesconi, K. A.; Thomas-Oates, J. E. Anal. Chem. 2000, 72, 357–366. (23) Infante, H. G.; O’Connor, G.; Rayman, M.; Hearn, R.; Cook, K. J. Anal. At. Spectrom. 2006, 21, 1256–1263. (24) Dernovics, M.; Lobinski, R. J. Anal. At. Spectrom. 2008, 23, 72–83. (25) Reyes, L. H.; Encinar, J. R.; Marchante-Gayon, J. M.; Alonso, J. I. G.; SanzMedel, A. J. Chromatogr., A 2006, 1110, 108–116. (26) Dernovics, M.; Garcia-Barrera, T.; Bierla, K.; Preud’homme, H.; Lobinski, R. Analyst 2007, 132, 439–449. (27) Montes-Bayon, M.; LeDuc, D. L.; Terry, N.; Caruso, J. A. J. Anal. At. Spectrom. 2002, 17, 872–879. (28) Vonderheide, A. P.; Mounicou, S.; Meija, J.; Henry, H. F.; Caruso, J. A.; Shann, J. R. Analyst 2006, 131, 33–40. (29) Gergely, V.; Montes-Bayon, M.; Fodor, P.; Sanz-Medel, A. J. Agric. Food Chem. 2006, 54, 4524–4530.
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Indeed, the incapacity of online HPLC-ESI Q-TOF MS/MS for the identification of selenium metabolites in a biological extract is best illustrated by a recent work, where more than 20 Se-species were found by HPLC-ICPMS, but only four of them were detected in the ESI-MS spectrum and finally only two novel species could be assigned.23 A higher mass accuracy and a larger intrascan dynamic range can be obtained by FTICR mass spectrometry,32 but applications to speciation of heteroatom-tagged metabolites have been limited to one work only concerning arsenic compounds.21 An alternative for the characterization of metabolomes can be the use of an electrostatic ion trap mass analyzer (Orbitrap) using fast Fourier transformation to obtain mass spectra.33 The Orbitrap mass analyzer was preceded by a linear ion trap used to selectively fill an intermediate ion storage device (C-trap) with ions of interest.34 The Orbitrap was shown to reach the 5 ppm mass accuracy with >95% probability at a dynamic range of more than 5000, which is at least an order of magnitude higher than typical values reported for time-of-flight instruments.35 Owing to the resolution of 30 000-60 000, the accurate mass of an ion could be determined when the signal was reliably distinguished from noise (S/Np-p > 2...3).19 A big advantage of the orbital mass analyzer is the capacity of multistage mass spectrometry (MSn) largely facilitating structure elucidation. The objective of this study was the development of a direct HPLC-MSn analytical method based on the use of an Orbitrap mass analyzer for the identification of Se-metabolites in Se-rich yeast used for food and feed supplementation. Hydrophilic interaction chromatography36 is proposed for the first time in order to achieve the online separation and desalting of hydrophilic Se-metabolites prior to electrospray ionization, the quantitative recovery being controlled using the parallel element-specific detection of selenium by ICPMS. An important part of the analytical strategy was the demonstration that all the Secompounds present in the analyzed sample were identified by the parallel HPLC-ICPMS using an orthogonal separation mechanism (anion-exchange) optimized for the highest resolution. EXPERIMENTAL SECTION Reagents and Standards. All the reagents and chromatographic eluents were purchased from the Sigma group (SigmaAldrich-Fluka-Riedel-de Hae¨n; St. Quentin Fallavier, France) except for acetonitrile that was obtained from Carlo Erba (RSplus grade; Milan, Italy). Deionized water (18.2 MΩ cm; Millipore; Guyancourt, France) was used throughout. Sample. The sample used as a model for this study was the most intense metabolite fraction isolated from a Se-rich yeast sample, Sel-Plex (Alltech, Nicholasville, KY) corresponding to a yeast strain Saccharomyces cerevisiae CNCM I-3060. The isolation (30) Ouerdane, L.; Mari, S.; Czernic, P.; Lebrun, M.; Lobinski, R. J. Anal. At. Spectrom. 2006, 21, 676–683. (31) Busto, M. E. D.; Montes-Bayon, M.; Blanco-Gonzalez, E.; Meija, J.; SanzMedel, A. Anal. Chem. 2005, 77, 5615–5621. (32) Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S. Mass Spectrom. Rev. 1998, 17, 1–35. (33) Makarov, A. Anal. Chem. 2000, 72, 1156–1162. (34) Schwartz, J. C.; Senko, M. W.; Syka, J. E. P. J. Am. Soc. Mass Spectrom. 2002, 13, 659–669. (35) Makarov, A.; Denisov, E.; Kholomeev, A.; Baischun, W.; Lange, O.; Strupat, K.; Horning, S. Anal. Chem. 2006, 78, 2113–2120. (36) Alpert, A. J. J. Chromatogr. 1990, 499, 177–196.
procedure based on aqueous extraction followed by size-exclusion chromatography was described elsewhere.37,38 Instrumentation. HPLC (hydrophilic interaction liquid chromatography (HILIC) or anion-exchange)-ICPMS coupling was achieved by using an Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA) connected to an Elan 6000 ICPMS (PE-Sciex, Ontario, Canada) for element-specific detection on 77Se, 82 Se, 88Sr, and 103Rh. For the HILIC-ESI-MS experiments, an Accela high speed liquid chromatography (Thermo Fisher Scientific, Waltham, MA) system was connected to a hybrid linear ion trap/Orbitrap mass analyzer (Thermo Fisher Scientific) used in either full-scan TOF mode or in product ion (MSn) mode. The HILIC-ESI-MS coupling was carried out via an Ion Max ESI electrospray ion source (Thermo Fisher Scientific). For sample digestion, a Multiwave 3000 microwave digestion system (Anton Paar; Courtaboeuf, France) was used. Procedures. Determination of the Total Selenium for the Column Recovery Measurements. A sample (or aliquots to analyze) was mixed with 4.0 mL of concentrated HNO3 and 4.0 mL of H2O2 in digestion tubes. The temperature was raised to 210 °C within 15 min and held for 20 min. The total Se concentration was determined (Elan 6000) using the 77Se and 82Se isotopes by the method of standard additions using Rh as an internal standard. Hydrophilic Interaction Liquid Chromatography. A TSK-Gel Amide80 (250 mm × 4.6 mm × 5 µm; Tosoh Biosciences, Stuttgart, Germany) HILIC column equipped with a matching guard column was used. Gradient elution (0.8 mL min-1) was carried out using eluent A, 0.1 v/v % trifluoroacetic acid (TFA) in acetonitrile, and eluent B, 0.1 v/v % TFA in water. The program was 0-4 min 97% A, 4-35 min up to 100% B, 35-45 min 100% B. The sample was dissolved in a 100 µL solution containing 97% of eluent A and 3% of eluent B. HILIC-Electrospray MSn. The injection volume was 30 µL. The ion source was operated in the positive ion mode at 4 kV. Capillary temperature was set to 300 °C. Nitrogen sheath gas was set to 15 units, while the auxiliary and sweep gases were set to 0 units. The resolving power of the Orbitrap (full width at half-height, fwhh) was set to nominal 60 000 (at m/z ) 400; 1 s scan cycle time) in full scan mode. For the MSn experiments, the product ions from the [M + H]+ charged target ions were generated in the LTQ trap at a collision energy setting of 35% and using an isolation width of 10 Da, in order to include and recognize the selenium isotopes in the fragments. The product ions were transferred to the Orbitrap part of the instrument for accurate mass measurement at a resolution of 7500 (fwhh) using internal mass-lock calibration based on the presence of system impurities of m/z ) 391.284 290 and 413.266 230 (dioctyl phthalate (DOP; [M + H]+) and [M + Na]+, respectively). The data recorded from either 100.00 (full scan mode) or 60.00 (MSn mode) to 1000.00 were processed with Xcalibur 2.0 software (Thermo Fisher Scientific). Initial calibration of the instrument was performed using the standard LTQ calibration mixture with caffeine and the peptide MRFA, dissolved in 50:50 v/v % water/acetonitrile solution. HILIC-ICPMS. The dissolved sample was diluted 20-fold with a solution containing 97% of eluent A and 3% of eluent B. The (37) Encinar, J. R.; Sliwka-Kaszynska, M.; Polatajko, A.; Vacchina, V.; Szpunar, J. Anal. Chim. Acta 2003, 500, 171–183. (38) Encinar, J. R.; Ouerdane, L.; Buchmann, W.; Tortajada, J.; Lobinski, R.; Szpunar, J. Anal. Chem. 2003, 75, 3765–3774.
injection volume was 200 µL. The column effluent was split 1:1 with a T-piece, and the flow was made up to 1.0 mL min-1 with 5 v/v % HNO3 solution delivered by a peristaltic pump at 0.6 mL min-1 prior to nebulization. This postcolumn split and makeup was necessary to compensate for the loss of sensitivity of Se detection due to the acetonitrile introduction into an ICP. Anion Exchange HPLC-ICPMS. A PRP-X100 strong anionexchange (SAX) column (250 mm × 4.1 mm × 10 µm; Hamilton, Reno, NV) was fitted with a matching guard column filled with the identical phase.24 Gradient elution was made with ammonium acetate (buffer A, 25 mM; buffer B, 250 mM; pH 5.5) delivered at 1.5 mL min-1. The program was 0-4 min 100% A, 4-40 min up to 100% B, 40-60 min 100% B. The sample was dissolved in buffer A. The injection volume was 10 µL. Accurate Mass Analysis and the Use of Elementary Calculator Tool (ECT). In order to assign the structure, a bottom-up approach was used. It started from the analysis of the MS4 data and continued with MS3, MS2, and MS. For all the calculations, the elementary calculator tool was set as follows: mass error [10 ppm) mass accuracy.24 As their intensities were adequate to carry out MS2, MS3, and MS4 experiments, the data of all the five species were evaluated. The complete structure identification approach is described in detail below for one of these species, that with m/z ) 604.081 79. The detailed data for the other four are shown in the Supporting Information and summarized in Table 1. The third group includes two species with low intrascan abundance (m/z ) 625.015 81 and m/z ) 652.026 31). Their intensities were too low for fragmentation studies; however, their structures could be assigned according to accurate mass information and analogy with the other, well-characterized species. Principle of Structure Assignment of a Se-Species via Accurate Mass and Collision Induced Dissociation (CID)-MSn in HILIC-LTQ Orbital Trap MS: Species with m/z ) 604.081 79. Figure 3 presents a cascade of MSn spectra taken at tR ) 17.60 min corresponding to the elution of the species with m/z ) 604.081 79 (monoisotopic mass of the M + H+ ion). The species shows the isotopic pattern characteristic of the presence of one Se atom. The approach to the structure identification developed here starts at the MS4 stage (Figure 3d). ECT gives only one hit for the elemental composition of the most abundant fragment present, C5H7N2O3+ [m/z ) 143.045 119; -1.483 ppm] and only one hit for the second most abundant one, C4H7N2O3+ [m/z ) 131.045 119; -2.318 ppm] (Figure 3d). This information, completed with the observation of a single Se-atom in the parent ion, points to only one possible elemental composition of the parent ion observed in the MS3 spectrum (m/z ) 256.949 34, Figure 3c); this is C5H9N2O3SSe+ [m/z ) 256.949 361; -0.079 ppm].
(44) Garcı´a-Reyes, J. F.; Dernovics, M.; Giusti, P.; Lobinski, R. J. Anal. At. Spectrom. 2006, 21, 655–665.
(45) McSheehy, S.; Pohl, P.; Szpunar, J.; Potin-Gautier, M.; Lobinski, R. J. Anal. At. Spectrom. 2001, 16, 68–73.
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Figure 1. (a-i) Full-scan mass spectra of the Se-species detected throughout the HILIC-LTQ/Orbitrap chromatogram. The insets show the scaled-up isotopic patterns referring to the presence of either one (a, c, e, g, and i) or two (b, d, f, and h) selenium atoms in the molecule. Note that eight of the the nine Se-species presented are supposed to form Se-S/Se-Se analogue couples. Analytical Chemistry, Vol. 80, No. 11, June 1, 2008
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Figure 2. (a) Extracted ion chromatograms for the Se-species detected throughout the chromatogram (cf. Figure 1). (b) HILIC-ICPMS chromatogram of the sample analyzed. The blue line shows the signal of 88Sr. (c) Anion-exchange HPLC-ICPMS chromatogram of the sample analyzed in the maximum resolution conditions.
The MS3 (Figure 3c) spectrum is rich in assignable fragments. Besides the one above, the two fragments in the low mass range (m/z ) 131.044 98 and m/z ) 143.044 97) are identical with those found on the MS4 stage. ECT provides a unique possible composition for the monoselenized fragment at m/z ) 224.977 16: C5H9N2O3Se+ [m/z ) 224.977 289; -0.586 ppm]. On the other hand, the elemental composition of the fragments at m/z ) 179.048 37 and at m/z ) 282.964 81 gives three and four possible hits by ECT and provides only circumstantial, however valuable, information. In turn, the composition of the known fragments on the MS3 stage helps to determine unambiguously the composition of their parent ion at m/z ) 345.997 25 as C8H16N3O5SSe+ [m/z ) 345.997 04; 0.621 ppm]. Now, formulas containing phosphorus or including more than eight carbon atoms can be eliminated for the unassigned ions of the MS3 level, allowing the assignment of the formula C5H11N2O3S+ to the m/z = 179.048 37 fragment [m/z ) 179.048 491; -0.672 ppm] and C7H11N2O3SSe+ to the m/z = 282.964 81 fragment [m/z ) 282.965 011; -0.700 ppm]. 3980
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For the two other abundant fragments in the MS2 spectrum (m/z ) 475.040 12 and m/z ) 586.072 90) (Figure 3b), the ECT gives too many possible compositions at the 5 ppm accuracy threshold. Therefore they cannot be directly used for the assignment of the genuine Se-species at m/z ) 604.081 79; for example, the ECT gives 15 hits on the basis of the composition of its daughter ion at m/z ) 345.997 25. At this level, the analysis of the (neutral) losses and the hypothesis of a glutathione conjugate give useful hints regarding the final structure: (1) the most abundant transition, m/z ) 604.081 79 f m/z ) 475.040 12 can be due to the characteristic loss of γ-Glu; (2) another transition m/z ) 604.081 79 f m/z ) 586.072 90 can be a characteristic loss of H2O from a free sCOOH group; (3) the third transition, m/z ) 604.081 79 f m/z ) 345.997 25 can be a characteristic loss of two γ-Glu residues; (4) the formation of the m/z ) 179.048 37 ion on the MS3 level fits the elemental composition of a cysteinyl-glycine moiety; (5) the formation of the m/z ) 224.977 16 ion on the MS3 level fits the elemental composition of a selenocysteinyl-glycine moiety. The summary of this characteristic spectral information excludes all formula of the m/z ) 604.081 79 Se-species with phosphorus and results in one ECT hit corresponding to the formula C18H30N5O11SSe+ [m/z ) 604.082 226; -0.727 ppm]. This elemental composition, the detected losses, and most of the fragments support the isomeric structures of either a conjugated selenoglutathione and a γ-glutamoyl-cysteine moiety or a conjugated glutathione and a γ-glutamoyl-selenocysteine moiety. Therefore, as shown in Figure 3, both versions of fragments are shown. Under physiological conditions, the sulfur analogue of this conjugate is formed by the activity of dipeptidases that can extracellularly remove the glycyl moiety from glutathione.46 Orbital Trap MSn vs Q-TOF MS for Speciation Analysis of Se-Metabolites. Figure 3 and Table 1 demonstrate the mass accuracies between 0.017-2.25 ppm measured for all the ions identified virtually regardless of the stage of MSn fragmentation. The mass accuracy of a large majority of measurements falls below the 1 ppm threshold. This mass accuracy is 1-2 orders of magnitude better than ESI-Q-TOF MS with similar sample introduction conditions. Consequently, it plays a key role in the correct assignment of the elemental composition by considerably limiting the number of possible ECT hits. Another important feature of the presented approach is the possibility to detect the isotopic pattern in the MS2-MS4 spectra due to the wide (10 Da) sampling window selected. In this way, the number of possible elemental formulas to be considered is reduced. This feature of the Orbitrap instrument was exploited in by Peterman et al.47 and Nielen et al.48 for chlorine-containing metabolites and has been applied here to Se for the first time. Note that a sampling window wider than 1.0 Da in the Q-TOF would have resulted either in an overwhelmed MS2 spectrum or in a spectrum poor in assignable fragments, depending on the collision energy settings. Without MS3 and MS4 data, none of them would have contributed to a successful structure elucidation. (46) Wang, W.; Ballatori, N. Pharmacol. Rev. 1998, 50, 335–356. (47) Peterman, S. M.; Duczak, N.; Kalgutkar, A. S.; Lame, M. E.; Soglia, J. R. J. Am. Soc. Mass Spectrom. 2006, 17, 363–375. (48) Nielen, M. W. F.; van Engelen, M. C.; Zuiderent, R.; Ramaker, R. Anal. Chim. Acta 2007, 586, 122–129.
Table 1. Detected and Assigned Fragments from the HILIC-LTQ-Orbitrap Analysis of the Four Se-Species Presented in Figure 1a-c,ha Se-species description conjugate of glutathione and 2,3-dihydroxy-propionyl-DHP selenocysteine (see Figure 4a and Figure S-1)
MSn experiment
C16H27N4O11SSe+
563.055 68
563.055 30
-0.675
MS2 MS2 MS2 MS3 MS3 MS3 MS3
1, loss of Gly 2, loss of γ-Glu 3, loss of glutathione 4, loss of 2,3-DHP 5, losses of Gly and H2O 6, loss of Cys-Gly 7, losses of formic acid, Cys-Gly and Se 8, loss of formic acid 9, loss of 2,3-DHP 10, losses of formic acid and Se entire molecule
C14H22N3O9SSe+ C11H20N3O8SSe+ C6H10NO5Se+ C8H16N3O5SSe+ C9H13N2O5SSe+ C6H10NO5Se+ C5H8NO3+
488.023 65 434.013 08 255.971 87 345.997 83 340.970 49 255.971 87 130.049 87
488.024 43 434.013 69 255.972 22 345.997 68 340.970 26 255.971 61 130.049 79
1.598 1.405 1.367 -0.434 -0.675 -1.016 -0.615
C5H8NO3Se+ C3H6NO2Se+ C5H8NO3+
209.966 39 167.955 83 130.049 87
209.966 26 167.955 57 130.049 73
-0.619 -1.518 -1.088
C17H29N4O11SSe+
577.071 33
577.071 23
-0.172
C15H24N3O9SSe+ C12H22N3O8SSe+ C7H12NO5Se+ C10H15N2O5SSe+ C7H12NO5Se+ C4H8NO2Se+
502.039 30 448.028 73 269.987 52 354.986 14 269.987 52 181.971 48
502.039 59 448.028 74 269.987 66 354.985 55 269.987 29 181.971 18
0.576 0.017 0.510 -1.668 -0.836 -1.648
C5H11N2O3S+
179.048 49
179.048 34
-0.837
C7H10NO4Se+ C6H10NO3Se+ C4H8NO2Se+ C3H6NSe+
251.976 96 223.982 04 181.971 48 135.966 00
251.976 70 223.982 10 181.971 25 135.965 88
-1.032 0.268 -1.264 -0.853
C16H27N4O11Se2+
611.000 13
610.999 76
-0.599
C14H22N3O9Se2+ C11H20N3O8Se2+ C6H10NO5Se2+
535.968 10 481.957 53 335.888 39
535.968 69 481.958 40 335.889 01
1.106 1.801 1.838
C6H10NO5Se2+
335.888 39
335.888 40
0.030
C6H8NO4Se2+
317.877 83
317.877 62
-0.645
C6H8NO5Se+
253.956 22
253.955 87
-1.370
C6H8NO4Se2+ C5H8NO3Se2+ C6H8NO5Se+
317.877 83 289.882 91 253.956 22
317.877 59 289.882 81 253.955 92
-0.728 -0.345 -1.177
C3H6NO2Se2+ C20H33N6O12Se2+
247.872 35 709.048 14
247.872 73 709.047 61
1.533 -0.745
C20H31N6O11Se2+ C15H26N5O9Se2+ C10H19N4O6Se2+
691.037 58 580.005 55 450.962 95
691.037 44 580.006 85 450.963 32
-0.203 2.250 0.816
C5H9N2O3Se2+
304.893 81
304.894 05
C10H19N4O6Se2+ C5H9N2O3Se2+
450.962 95 304.893 81
450.962 56 304.893 17
-0.868 -2.107
C5H9N2O3Se2+
304.893 81
304.893 37
-1.428
MS1
MS2 MS2 MS2 MS3 MS3 MS3
MS4 MS4 MS4 MS4 MS1
MS2 MS2 MS2 MS3 MS3 MS3 MS4 MS4 MS4
conjugate of two selenoglutathione moieties (see Figure 4d and Figure S-4)
theoretical mass, measured mass, difference, m/z m/z ppm
entire molecule
MS3
conjugate of seleno-glutathione and 2,3-dihydroxy-propionylselenocysteine (see Figure 4c and Figure S-3)
elemental composition
MS1
MS4 MS4 MS4 conjugate of glutathione and 2,3-dihydroxy-propionylseleno-homocysteine (see Figure 4b and Figure S-2)
descriptions of detected ions and fragments
MS4 MS1 MS2 MS2 MS2 MS2 MS3 MS3 MS4
1, loss of Gly 2, loss of γ-Glu 3, loss of glutathione 4, losses of Gly and H2O 5, loss of Cys-Gly 6, losses of Cys-Gly and 2,3-DHP 7, loss of 2,3-DHP-selenohomocysteine 8, loss of H2O 9, loss of formic acid 10, loss of 2,3-DHP 11, losses of formic acid and 2,3-DHP entire molecule
1, loss of Gly 2, loss of γ-Glu 3, loss of heteroelementfree glutathione 4, loss of heteroelementfree Cys-Gly 5, losses of heteroelementfree Cys-Gly and H2O 6, loss of SeCys-Gly and double bond formation 7, loss of H2O 8, loss of formic acid 9, loss of Se and double bond formation 10, loss of 2,3-DHP entire molecule 1, loss of H2O 2, loss of γ-Glu 3, losses of two γ-Glu moieties 4, losses of γ-Glu and heteroelementfree glutathione 5, loss of γ-Glu 6, loss of heteroelementfree glutathione 7, loss of heteroelementfree Cys-Gly
0.787
a The proposed structures and fragmentation pathways are shown in Figure 4. All the indicated mass losses relate to a given MSn experiment where the ions with masses in bold were fragmented. The corresponding high resolution full-scan and fragmentation spectra are given in the Supporting Information (SI).
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Figure 3. (a) Mass spectrum taken at tR ) 17.60 min showing a species at m/z ) 604.081 79. (b-d) Collision induced dissociation (CID)-MS2, MS3, and MS4 spectra of the Se-species detected. The arrows indicate the parent ion selection for the subsequent MSn experiment. Ions are labeled with the molecular mass measured, elemental formula assigned, and the mass difference in ppm between the measured and theoretical masses. The numbers in circles denote to the proposed fragmentation events and resulting fragments (cf. Figure 3e-g). Annotated (′) numbers indicate the same fragment arising in subsequent CID steps. (e) Proposed structure for the detected species: conjugate of either selenoglutathione and γ-glutamoyl-cysteine or glutathione and γ-glutamoyl-selenocysteine. (e-g) Proposed fragmentation pathways for the detected fragments in the MS2, MS3, and MS4 experiments, respectively.
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Figure 4. (a-d) Proposed structures and fragmentation pathways of the Se-species detected at m/z ) 563.055 30 (cf. Figure 1a), m/z ) 577.071 23 (cf. Figure 1c), m/z ) 610.999 76 (cf. Figure 1b), and m/z ) 709.047 61 (cf. Figure 1h), respectively. The numbers in circles denote the ions discussed in Table 1.
Structure Assignments of the Other Detected Se-Species via Accurate Mass and CID-MSn. The approach outlined above has been successfully used for the elucidation of the other four compounds (m/z ) 577, 611, 563, and 709) which produced the
complete set of mass spectra down to MS4. The data are summarized in Table 1 whereas Figure 4shows the proposed structures. The Supporting Information presents the complete set of the mass spectra obtained. Analytical Chemistry, Vol. 80, No. 11, June 1, 2008
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Table 2. Proposed Elemental Compositions and Structures of the Se-Species presented in Figure 1d (m/z ) 625.015 81) and Figure 1f (m/z ) 652.026 31)a
a
For details, see text.
Structure Assignments for Minor Species via Accurate Mass and Correlations with More Abundant Species. The S/N ratios for the two remaining compounds (m/z ) 625 and 652, parts d and f of Figure 1, respectively) were found too low to allow meaningful fragmentation. However, the presence of the doubly selenized isotopic patterns of the species allows a hypothesis to be put forward for this species to be the Se-Se analogues of the previously identified species at m/z ) 577 (cf. Figure 1c) and m/z ) 604 (cf. Figure 1e) containing a Se-S bridge. This hypothesis is validated by the accurate mass measurements (Table 2). Indeed, for the m/z ) 625.015 81 compound, the proposed empirical formula fits within 0.048 ppm of the theoretical value whereas for the m/z ) 652.026 31 compound within 0.568 ppm. CONCLUSIONS The coupling of HILIC chromatography with the hybrid LIT/ Orbitrap system allows the direct comprehensive speciation of selenium in conditions where the low intrascan dynamic range of Q-TOF MS does not either allow the detection of the pseudomolecular ion or produces very low mass accuracy (>50 ppm). The 1-2 ppm and sub-ppm mass accuracy is obtained on a routine
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basis for a real-world sample and is hardly affected by the species concentration. A further incontestable advantage over Q-TOF is this accuracy being preserved in mass spectra up to MS4 while the large fragmentation window allows following the lineage over the whole isotopic pattern range. When completed by the elemental mass flow monitoring and chromatographic recovery by ICPMS, the technique seems to be currently an ultimate tool for exhaustive quantitative heteroatom-tagged metabolomics. ACKNOWLEDGMENT M.D. acknowledges a Marie-Curie Fellowship (Grant MERG-CT2006-044951). The authors thank Eric Génin (Thermo France) for his help with the analyses, Dr. Laurent Ouerdane for valuable discussions, and Dr. Gérard Bertin for providing the Se-rich yeast sample. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs. org. Received for review January 28, 2008. Accepted March 21, 2008. AC8002038
