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Anal. Chem. 2010, 82, 5887–5891

A Strategy for the Determination of the Elemental Composition by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Based on Isotopic Peak Ratios Daisuke Miura,† Yukiko Tsuji,‡ Katsutoshi Takahashi,†,‡,§ Hiroyuki Wariishi,*,†,‡,| and Kazunori Saito⊥ Innovation Center for Medical Redox Navigation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 12-8582, Japan, Bio-Architecture Center, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan, Research Institute of Instrumentation Frontier, National Institute of Advanced Industrial Science and Technology, 2-42 Aomi, Koutou-ku, Tokyo, Japan, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan, and Bruker Daltonics K.K., 3-9, B-6F, Moriya-cho, Kanagawa, Yokohama, Kanagawa 221-0022, Japan We propose a novel strategy for determining the elemental composition of organic compounds using the peak ratio of isotopic fine structure observed by high-magnetic field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Using 3′-phosphoadenosine 5′phosphosulfate and CTU guanamine as standard organic compounds, isotopic peaks derived from 15N-, 34S-, and 18 O-substituted forms were separated from 13C-substituted species. Furthermore, these isotopic peaks were quantitatively detected and closely matched the natural abundance of each element. These data successfully led us to determine the one elemental composition in a standard independent manner. The approach should be particularly amenable to the metabolomics research field. One of the goals of metabolomics research is to obtain structural information about every low-molecular weight component in an organism.1,2 While the identification of chemical structures of metabolites is important, the structural identification of organic substances such as natural products or artificial chemicals is also a key strategy for further functional and application studies. The most common approach for structure determination of small molecules combines 1H and 13C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). However, the rapid annotation and structural elucidation of small molecules remains a challenge for complex mixtures such as biofluids or extracts taken from biological samples. Recent interest in metabolomics and safety evaluation techniques highlights the decisive sensitivity and throughput * To whom correspondence should be addressed. Telephone/Fax: +81-92642-2992. E-mail: [email protected]. † Innovation Center for Medical Redox Navigation, Kyushu University. ‡ Bio-Architecture Center, Kyushu University. § National Institute of Advanced Industrial Science and Technology. | Faculty of Agriculture, Kyushu University. ⊥ Bruker Daltonics K.K. (1) Kind, T.; Fiehn, O. BMC Bioinf. 2007, 8, 105. (2) Kind, T.; Fiehn, O. BMC Bioinf. 2006, 7, 234. 10.1021/ac902931x  2010 American Chemical Society Published on Web 06/03/2010

advantage of MS over NMR.3 Library or database search strategies using the exact MS and MS/MS pattern of mass spectra of known and available compounds are well-established chemical annotations with MS.4,5 This strategy is not a datadriven method but is completely dependent on the spectral databases or comparisons with reference authentic standard spectra, despite the fact that commercially available compounds are within 20% of whole biological metabolites.6 The elemental composition represents one of the most important pieces of information for the determination of the structure of completely unknown substances. Although MS is highly sensitive and an easyto-use technique that provides indispensable mass data for substances, the acquisition of only the monoisotopic mass cannot always identify unknown candidates even if high-mass accuracy instruments are used. Even though some kinds of software use the abundances of the other isotopes in a nominally resolved cluster for elemental composition estimation, they cannot sufficiently narrow the candidates because these peaks are a summation of the individual components with the same nominal mass. In this case, the intensities of these isotopic peaks mainly reflect the amounts of abundant isotopes such as 13C and 34S, and the direct confirmation of the existence of other elements is difficult. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is well recognized as a MS method with the highest mass accuracy (within 1 ppm).7 However, a series of candidates (3) Southam, A. D.; Payne, T. G.; Cooper, H. J.; Arvanitis, T. N.; Viant, M. R. Anal. Chem. 2007, 79, 4595–4602. (4) Wishart, D. S.; Tzur, D.; Knox, C.; Eisner, R.; Guo, A. C.; Young, N.; Cheng, D.; Jewell, K.; Arndt, D.; Sawhney, S.; Fung, C.; Nikolai, L.; Lewis, M.; Coutouly, M. A.; Forsythe, I.; Tang, P.; Shrivastava, S.; Jeroncic, K.; Stothard, P.; Amegbey, G.; Block, D.; Hau, D. D.; Wagner, J.; Miniaci, J.; Clements, M.; Gebremedhin, M.; Guo, N.; Zhang, Y.; Duggan, G. E.; Macinnis, G. D.; Weljie, A. M.; Dowlatabadi, R.; Bamforth, F.; Clive, D.; Greiner, R.; Li, L.; Marrie, T.; Sykes, B. D.; Vogel, H. J.; Querengesser, L. Nucleic Acids Res. 2007, 35, D521–D526. (5) Horai, T.; Arita, M.; Nishioka, T. 1st International Conference on BioMedical Engineering and Informatics (BMEI 2008), Sanya, Hainan, China, 2008. (6) Soga, T.; Ohashi, Y.; Ueno, Y.; Naraoka, H.; Tomita, M.; Nishioka, T. J. Proteome Res. 2003, 2, 488–494.

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Figure 1. Result of the simulation for (A) the number of candidate formulas vs the observed mass to charge ratio at various mass accuracies and (B) isotopic peak appearance at various mass resolutions. (A) The elemental composition of the candidate was calculated under the limitation of the number of each element (C0-200H0-500N0-20O0-20S0-5P0-5). The number of candidate formulas was plotted for each tolerance of mass spectral measurement (100-0.01 mDa). (B) The shape of the second isotopic peak [(M - H+ + 2)-] of ATP in negative ion mode under different mass resolution was simulated by using DataAnalysis version 3.4.

consisting of CHONPS elements are found in a monoisotopic mass peak measured for a low-molecular weight compound with this mass accuracy. For example, for an instrument with a 0.1 mDa mass accuracy, more than eight possible candidates arise for a species with an ion at m/z 400 (Figure 1A). Thus, the analysis of a monoisotopic mass with a highly accurate MS instrument alone presents difficulties in determining the elemental composition. This is also the case when empirical rules such as the N-rule and electron configuration and/or elemental restrictions obtained from other analytical methods are used. In contrast, FT-ICR-MS provides not only 12 T are commercially available, and a full width at half-maximum (fwhm) resolving power of >500000 can be obtained even in a broadband mode, although time-of-flight MS can be achieved within a fwhm of 50000. Theoretically, isotopic fine structures derived from 15N, 34S, and 18O are clearly resolved from the isotopic peaks from carbon under more than 300000 fwhm (Figure 1B, as is the case for ATP), and the ratio of these peaks will be dependent on the natural abundance of the isotopes for each element. In this note, we extend a prior strategy8 for elemental composition determination of CHONPS (organic) compounds by ultrahigh mass resolution analysis with high-magnetic field FTICR-MS. This technique may represent a basic strategy that can be employed in the research fields of natural products, artificial chemicals, and metabolomics. (7) Marshall, A. G.; Hendrickson, C. L. Annu. Rev. Anal. Chem. 2008, 1, 579– 599. (8) Shi, D. S.; Hendrickson, C. L.; Marshall, A. G. Proc. Natl. Acad. Sci. U.S.A. 1998, 95 (20), 11532–11537.

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MATERIALS AND METHODS Reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan) with the exception of 3′-phosphoadenosine 5′-phosphosulfate (PAPS) which was purchased from Calbiochem (San Diego, CA). Deionized water was obtained from a Milli-Q system (Millipore). An Apex-Q120e FT-ICR-MS instrument (Bruker Daltonics, Inc., Billerica, MA) was used for MS measurements. The MS system was equipped with a 12 T superconducting magnet (Magnex Scientific, Oxford, U.K.). A quadrupole mass isolator was set at the front of the ICR analyzer to enhance the dynamic range of the target peaks. An Apollo II electrospray ionization (ESI)/matrixassisted laser desorption ionization (MALDI) dual ion source (Bruker Daltonics, Inc.) was used in ESI mode. This ion source has dual ion funnels, and ions were orthogonally injected through a glass capillary between the first funnel and deflector plate. The spray shield voltage was set to -4.0 kV, and the capillary entrance was set to -4.5 kV in the positive ion mode. The sprayer was grounded (0 V). The dry gas temperature was set to 150 °C. The nebulizing gas pressure was set to 1.0 MPa. Samples were injected using a syringe pump, and the flow rate was 120 µL/h. Samples were dissolved in a solvent (49:49:2 H2O/MeOH/acetic acid mixture in positive ion mode and 50:50 H2O/MeOH mixture in negative ion mode) to give 100 fmol/µL. Ions were initially trapped in the second storage/collision cell hexapole through the quadrupole to enhance the ion population and then injected into the ICR cell. The ion storage time in this hexapole was regulated to control the number of the ions. The difficulty in the quantification of the elements was raised in this study, especially because of the space charge. Nonideal ion motions were observed when the number of ions injected into the ICR cell was increased, and the resultant quantification of the

Figure 2. Mass spectral observation of CTU guanamine (C17H26N10O4) in broadband mode by using high-magnetic field FT-ICR-MS. The ionization source was operated in ESI positive ion mode. Multiplet isotopic peaks were observed in the (M - H+ + 1)- and (M - H+ + 2)regions, and these peaks were successfully assigned to the substitution of a stable isotope for each element.

isotope peaks was challenging. As previously reported,9-11 when the ions are trapped in the ICR cell at a high density, ion-ion interactions are induced and even peak coalescence is observed. Consequently, to suppress the space charge effect, the number of ions injected into the ICR cell must be limited. In this study, the number of the ions was reduced to as few as possible by changing the ion accumulation time in the hexapole (typically 10-100 ms). This ensured that the maximum error was 10%. MS spectra were recorded using apexControl version 2.0 (Bruker Daltonics, Inc.), and the acquired data were processed using DataAnalysis version 3.4 (Bruker Daltonik, GmbH, Bremen, Germany) to pick peaks and calculate peak areas. Calculation of masses and isotope abundances was based on the data from the National Institute of Standards and Technology (Gaithersburg, MD).12 The elemental composition of the candidate for the observed mass was calculated by the combination of C, H, N, O, P, and S elements under the limitation of the number of each element (C0-200H0-500N0-20O0-20S0-5P0-5). For example, eight possible candidates arise for a species with an ion at m/z 400-401 for an instrument with a 0.05 mDa mass accuracy. RESULTS AND DISCUSSION Several projects that focused on the identification of biological metabolites using mass spectrometric information have been reported.13-15 In principle, one elemental composition shows one Naito, Y.; Inoue, M. Int. J. Mass Spectrom. 1996, 157/158, 85–96. Mitchell, D. W.; Smith, R. D. Phys. Rev. E 1995, 52, 4366–4386. Mitchell, D. W.; Smith, R. D. J. Mass Spectrom. 1996, 31, 771–790. http://physics.nist.gov/PhysRefData/Compositions/index.html. Fiehn, O.; Kopka, J.; Trethewey, R. N.; Willmitzer, L. Anal. Chem. 2000, 72, 3573–3580. (14) Iijima, Y.; Nakamura, Y.; Ogata, Y.; Tanaka, K.; Sakurai, N.; Suda, K.; Suzuki, T.; Suzuki, H.; Okazaki, K.; Kitayama, M.; Kanaya, S.; Aoki, K.; Shibata, D. Plant J. 2008, 54, 949–962. (15) Scalbert, A.; Brennan, L.; Fiehn, O.; Hankemeier, T.; Kristal, B. S.; van Ommen, B.; Pujos-Guillot, E.; Verheij, E.; Wishart, D.; Wopereis, S. Metabolomics 2009, 5, 435–458. (9) (10) (11) (12) (13)

molecular weight and one molecular weight determines one elemental composition; however, the relationship strongly depends on the accuracy of the molecular weight measurement. Figure 1A shows the number of elemental composition candidates consisting of CHONPS elements for organic substances based on a series of MS accuracies. For the ion at m/z 500, more than 1000 candidates were found at a 10 mDa accuracy, and more than 20 candidates were identified at a 0.1 mDa accuracy. The highest mass accuracy can be obtained using a FT-ICR-MS instrument with an ultrahigh magnetic field; however, even with a mass accuracy of 0.05-0.1 mDa, there were still several candidates. Thus, it is difficult to determine the elemental composition of metabolites using only an accurate m/z value. The mass spectral observation of both the presence and relative abundance of isotopic peaks should provide indispensable information for the determination of the elemental composition of metabolites if the isotopic peaks derived from each element can be measured quantitatively. To validate the quantitative performance of FT-ICR-MS for detection of isotopic fine structure, CTU guanamine (C17H26N10O4) was first subjected to the measurement in ESI positive ion mode with a mass range from m/z 400 to 3000. The protonadducted molecular ion (M + H+) was observed at m/z 435.22111 (error of -0.02 ppm) with a resolving power of 300000 fwhm. Two isotopic peaks existing as multiplet peaks were observed in the regions of m/z 436.2 and 437.2 (Figure 2). The isotopic peak at m/z 436.2 was a doublet. Using the difference in their exact masses from the monoisotopic peak, the individual components of the isotopic fine structure were assigned as C17H27N915N1O4 (m/z 436.21819, error of -0.1 ppm) and C1613C1H27N10O4 (m/z 436.22450, error of 0.16 ppm). The three components of the triplet isotopic peak found at m/z 437.2 were assigned as C1613C1H27N915N1O4 (m/z 437.22152, error of 0.2 ppm), C17H27N10O318O1 (m/z 437.22540, error of -0.07 ppm), and C1513C2H27N10O4 (m/z 437.22784, error of 0.02 ppm). These Analytical Chemistry, Vol. 82, No. 13, July 1, 2010

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Table 1. Peak Area Ratios of Each Isotopic Peak to Monoisotopic Peak in CTU Guanamine elemental composition for each peak

ratio for the measured area (%)

ratio for the simulated area (%)

C17H27N10O4 C17H27N915N1O4 C1613C1H27N10O4 C1613C1H27N915N1O4 C17H27N10O318O1 C1513C2H27N10O4

100.0 3.8 18.6 0.6 0.8 1.6

100.0 3.7 18.4 0.7 0.8 1.6

Table 2. Numbers of Each Element Calculated from Measured Peak Areas in CTU Guanamine element

calculated number

actual number

C N O

17.25 10.46 3.80

17 10 4

results indicate that FT-ICR-MS can clearly separate several isotopic peaks caused by isotopic substitution. Furthermore, the difference in atomic mass units for the 15N-substituted peak was clearly separated by 0.00631 Da (theoretically, 0.00632 Da) from the 13C-substituted peak. The 18O-substituted peak was also separated by 0.00244 Da (theoretically, 0.00246 Da) from the 13C2-substituted peak. The observed peak area ratios were then compared with those of the simulated spectrum on the basis of the natural abundance of the isotopes (Table 1). These results indicate that all peak areas for isotopic fine structure agree well with the simulated data. To estimate the numbers of the elements from the measured mass spectrum, each peak area of the isotopic peaks was divided by the peak area of the monoisotopic peak (Table 2). The numbers of elements can be calculated when the peak area ratios of each peak are divided by the natural abundances of the each element. The result of the calculated number of the elements was concordant with the actual value, and the maximum error among the three elements was that for nitrogen, with a value of 4.6%. These data suggest that the number of elements of carbon, nitrogen, and oxygen can be determined approximately by the method presented here [peak heights in FT-ICR MS are valid to 1-2% at best (and less accurately at lower signal-to-noise ratios)]. A large number of bioorganic substances consist of S and P in addition to the standard elements C, H, O, and N. 34S is a naturally stable isotope of 32S with a natural abundance of 4.29%, which gives rise to a relatively strong isotopic peak derived from the isotope substitution. Furthermore, the differences in atomic mass units for the 34S-substituted peak and 13C2- and 18 O-substituted peaks are 0.01091 and 0.00845 Da, respectively, which provides a sufficiently large mass difference for the separation of these isotopic peaks. In contrast, phosphorus has no natural isotopes; thus, the number of phosphorus atoms cannot be estimated directly from the isotopic fine structure. The following is a calculation algorithm used. (i) Find a monoisotopic peak. (ii) Assign the isotopic peaks originating from 15N, 13C, 34S, and 18O substitution by the mass difference between the monoisotopic mass and each peak. For example, the mass difference between 13C and the monoisotopic peak is 1.0034 Da. (iii) Calculate the ratio of a monoisotopic to isotopic 5890

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peak areas. (iv) Divide the values obtained in step iii by the ratio of natural abundances. The number of the four elements can be calculated when the isotopic peaks are separated from each other. (v) Multiply the values by a safety factor (tolerance of maximum error rate for estimation of the number of each element). (vi) Calculate the elemental composition with the constraints obtained in step v. For the verification of the algorithm for the determination of the elemental composition by FT-ICR-MS measurements, 3′-phosphoadenosine 5′-phosphosulfate (PAPS, C10H15O13N5P2S) was measured in ESI negative ion mode. In this algorithm, the numbers of C, H, N, O, and S were utilized as a constraint in the calculation of the elemental composition. Figure 3 shows the spectrum of PAPS with a resolving power of more than 600000 fwhm, and the deprotonated (M - H+)- peak was observed at m/z 505.97705. Triplet isotopic fine structure was observed in the (M - H+ + 1)- region, and the 15N-, 33S-, and 13C-substituted peaks were estimated from their exact mass. Triplet isotopic fine structure was also observed in the (M - H+ + 2)- region. The clear 34 S-derived isotopic peak was observed; however, the 33Sderived isotopic peak was a very minor species because the natural abundance of this isotope is 0.762%. The C9.8N4.5O12.7S1.0 composition (C10N5O13S for PAPS) was obtained by calculation of the number of each element. A maximum error of 10% was found for nitrogen. A safety factor has to be set in the calculation based on the number of elements that can be used as good constraints for the elemental composition analysis. Since the observed maximum error was 10% in the case of PAPS, the safety factor should be more than 10%. Therefore, a safety factor of 25% was used in these experiments. By multiplying the obtained constraints by the safety factor of 25%, we obtained the C8-13N4-6O10-16S1 composition. Taking into consideration the constraints obtained above, we can successfully narrow the candidate to only one elemental composition (C10H15O13N5P2S1). In contrast, 156 candidates (for C∞H∞N∞O∞S0-5P0-5) were calculated from information obtained for the exact monoisotopic mass (m/z 505.97905) with a mass tolerance of 0.5 mDa (1 ppm). In this study, quantification of the elements was conducted using the information about the resolved monoisotopic and isotopic peak areas with a maximum error of 10%. Both the ratios of each observed isotopic peak to the monoisotopic peak areas and the calculated number of each element were in good agreement with the results of the simulation. This indicates that ultrahigh resolution FT-ICR-MS has sufficient performance for the quantitative detection of peak areas and the ratio-based determination of the number of each element. Other constraints, such as the nitrogen rule, the known ratios of carbon to hydrogen, and/ or other elements and electron configuration, may aid in reducing the number of candidates. By using FT-ICR-MS, a higher resolving power is easily obtained for smaller molecules when compared the powers of other MS methods such as time-of-flight MS. Since a higher resolving power of more than 300000 is mandatory for the determination of the elemental composition described herein, smaller molecules,