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Spectral trees as a robust annotation tool in LC–MS based metabolomics

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Abstract

The identification of large series of metabolites detectable by mass spectrometry (MS) in crude extracts is a challenging task. In order to test and apply the so-called multistage mass spectrometry (MSn) spectral tree approach as tool in metabolite identification in complex sample extracts, we firstly performed liquid chromatography (LC) with online electrospray ionization (ESI)–MSn, using crude extracts from both tomato fruit and Arabidopsis leaf. Secondly, the extracts were automatically fractionated by a NanoMate LC-fraction collector/injection robot (Advion) and selected LC-fractions were subsequently analyzed using nanospray-direct infusion to generate offline in-depth MSn spectral trees at high mass resolution. Characterization and subsequent annotation of metabolites was achieved by detailed analysis of the MSn spectral trees, thereby focusing on two major plant secondary metabolite classes: phenolics and glucosinolates. Following this approach, we were able to discriminate all selected flavonoid glycosides, based on their unique MSn fragmentation patterns in either negative or positive ionization mode. As a proof of principle, we report here 127 annotated metabolites in the tomato and Arabidopsis extracts, including 21 novel metabolites. Our results indicate that online LC–MSn fragmentation in combination with databases of in-depth spectral trees generated offline can provide a fast and reliable characterization and annotation of metabolites present in complex crude extracts such as those from plants.

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References

  • Bedair, M., & Sumner, L. W. (2008). Current and emerging mass-spectrometry technologies for metabolomics. TrAC - Trends in Analytical Chemistry, 27, 238–250.

    Article  CAS  Google Scholar 

  • Böttcher, C., Roepenack-Lahaye, E. V., Schmidt, J., Schmotz, C., Neumann, S., Scheel, D., et al. (2008). Metabolome analysis of biosynthetic mutants reveals a diversity of metabolic changes and allows identification of a large number of new compounds in Arabidopsis. Plant Physiology, 147, 2107–2110.

    Article  PubMed  Google Scholar 

  • Clifford, M. N., Knight, S., & Kuhnert, N. (2005). Discriminating between the six isomers of dicaffeoylquinic acid by LC–MSn. Journal of Agricultural and Food Chemistry, 53, 3821–3832.

    Article  PubMed  CAS  Google Scholar 

  • De Vos, R. C. H., Moco, S., Lommen, A., Keurentjes, J. J. B., Bino, R. J., & Hall, R. D. (2007). Untargeted large-scale plant metabolomics using liquid chromatography coupled to mass spectrometry. Nature Protocols, 2, 778–791.

    Article  PubMed  Google Scholar 

  • Dunn, W.B. (2008). Current trends and future requirements for the mass spectrometric investigation of microbial, mammalian and plant metabolomes. Physical Biology, 5(1), 011001.

    Google Scholar 

  • Ferreres, F., Taveira, M., Pereira, D. M., Valentão, P., & Andrade, P. B. (2010). Tomato (Lycopersicon esculentum) seeds: New flavonols and cytotoxic effect. Journal of Agricultural and Food Chemistry, 58, 2854–2861.

    Article  PubMed  CAS  Google Scholar 

  • Francisco, M., Moreno, D. A., Cartea, M. E., Ferreres, F., García-Viguera, C., & Velasco, P. (2009). Simultaneous identification of glucosinolates and phenolic compounds in a representative collection of vegetable Brassica rapa. Journal of Chromatography A, 1216, 6611–6619.

    Article  PubMed  CAS  Google Scholar 

  • Gómez-Romero, M., Segura-Carretero, A., & Fernández-Gutiérrez, A. (2010). Metabolite profiling and quantification of phenolic compounds in methanol extracts of tomato fruit. Phytochemistry, 71, 1848–1864.

    Article  PubMed  Google Scholar 

  • Hooft, J. J. J.v.d., Vervoort, J., Bino, R. J., Beekwilder, J., & Vos, C. H. R.d. (2011). Polyphenol identification based on systematic and robust high-resolution accurate mass spectrometry fragmentation. Analytical Chemistry, 83, 409–416.

    Article  PubMed  Google Scholar 

  • Iijima, Y., Suda, K., Suzuki, T., Aoki, K., & Shibata, D. (2008). Metabolite profiling of chalcones and flavanones in tomato fruit. Journal of the Japanese Society for Horticultural Science, 77, 94–102.

    Article  CAS  Google Scholar 

  • Kind, T.M., & Fiehn, O. (2007). Seven golden rules for heuristic filtering of molecular formulas obtained by accurate mass spectrometry. BMC Bioinformatics, 8, 105.

    Google Scholar 

  • Kind, T., & Fiehn, O. (2010). Advances in structure elucidation of small molecules using mass spectrometry. Bioanalytical Reviews, 2, 23–60.

    Article  PubMed  Google Scholar 

  • Ma, Y. L., Cuyckens, F., Van den Heuvel, H., & Claeys, M. (2001). Mass spectrometric methods for the characterisation and differentiation of isomeric O-diglycosyl flavonoids. Phytochemical Analysis, 12, 159–165.

    Article  PubMed  CAS  Google Scholar 

  • Makarov, A., Denisov, E., Kholomeev, A., Balschun, W., Lange, O., Strupat, K., et al. (2006). Performance evaluation of a hybrid linear ion trap/Orbitrap mass spectrometer. Analytical Chemistry, 78, 2113–2120.

    Article  PubMed  CAS  Google Scholar 

  • Makarov, A., & Scigelova, M. (2010). Coupling liquid chromatography to Orbitrap mass spectrometry. Journal of Chromatography A, 1217, 3938–3945.

    Article  PubMed  CAS  Google Scholar 

  • Matsuda, F., Yonekura-Sakakibara, K., Niida, R., Kuromori, T., Shinozaki, K., & Saito, K. (2009). MS/MS spectral tag-based annotation of non-targeted profile of plant secondary metabolites. Plant Journal, 57, 555–577.

    Article  PubMed  CAS  Google Scholar 

  • Millán, S., Sampedro, M. C., Gallejones, P., Castellón, A., Ibargoitia, M. L., Goicolea, M. A., et al. (2009). Identification and quantification of glucosinolates in rapeseed using liquid chromatography–ion trap mass spectrometry. Analytical and Bioanalytical Chemistry, 394, 1661–1669.

    Article  PubMed  Google Scholar 

  • Moco, S., Bino, R. J., De Vos, R. C. H., & Vervoort, J. (2007a). Metabolomics technologies and metabolite identification. TrAC - Trends in Analytical Chemistry, 26, 855–866.

    Article  CAS  Google Scholar 

  • Moco, S., Bino, R. J., Vorst, O., Verhoeven, H. A., de Groot, J., van Beek, T. A., et al. (2006). A liquid chromatography-mass spectrometry-based metabolome database for tomato. Plant Physiology, 141, 1205–1218.

    Article  PubMed  CAS  Google Scholar 

  • Moco, S., Capanoglu, E., Tikunov, Y., Bino, R. J., Boyacioglu, D., Hall, R. D., et al. (2007b). Tissue specialization at the metabolite level is perceived during the development of tomato fruit. The Journal of Experimental Botany, 58, 4131–4146.

    Article  CAS  Google Scholar 

  • Olsen, H., Aaby, K., & Borge, G. I. A. (2009). Characterization and quantification of flavonoids and hydroxycinnamic acids in curly kale (Brassica oleracea L. convar. acephala var. sabellica) by HPLC-DAD-ESI-MSn. Journal of Agricultural and Food Chemistry, 57, 2816–2825.

    Article  PubMed  CAS  Google Scholar 

  • Rasche, F., Svatos, A., Maddula, R. K., Böttcher, C., & Böcker, S. (2011). Computing fragmentation trees from tandem mass spectrometry data. Analytical Chemistry, 83, 1243–1251.

    Article  PubMed  CAS  Google Scholar 

  • Rochfort, S. J., Imsic, M., Jones, R., Trenerry, V. C., & Tomkins, B. (2006). Characterization of flavonol conjugates in immature leaves of pak choi [Brassica rapa L. Ssp. chinensis L. (Hanelt.)] by HPLC–DAD and LC–MS/MS. Journal of Agricultural and Food Chemistry, 54, 4855–4860.

    Article  PubMed  CAS  Google Scholar 

  • Rochfort, S. J., Trenerry, V. C., Imsic, M., Panozzo, J., & Jones, R. (2008). Class targeted metabolomics: ESI ion trap screening methods for glucosinolates based on MSn fragmentation. Phytochemistry, 69, 1671–1679.

    Article  PubMed  CAS  Google Scholar 

  • Roepenack-Lahaye, E.v., Degenkolb, T., Zerjeski, M., Franz, M., Roth, U., Wessjohann, L., et al. (2004). Profiling of Arabidopsis secondary metabolites by capillary liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry. Plant Physiology, 134, 548–559.

    Article  Google Scholar 

  • Scigelova, M., & Makarov, A. (2006). Orbitrap mass analyzer—overview and applications in proteomics. Proteomics, 1, 16–21.

    Article  CAS  Google Scholar 

  • Sheldon, M. T., Mistrik, R., & Croleya, T. R. (2009). Determination of ion structures in structurally related compounds using precursor ion fingerprinting. Journal of the American Society for Mass Spectrometry, 20, 370–376.

    Article  PubMed  CAS  Google Scholar 

  • Slimestad, R., & Verheul, M. (2009). Review of flavonoids and other phenolics from fruits of different tomato Lycopersicon esculentum Mill. cultivars. Journal of Science Food Agriculture, 89, 1255–1270.

    Article  CAS  Google Scholar 

  • Sumner, L. W., Amberg, A., Barrett, D., Beale, M. H., Beger, R., Daykin, C. A., et al. (2007). Proposed minimum reporting standards for chemical analysis. Metabolomics, 3, 211–221.

    Article  CAS  Google Scholar 

  • Tohge, T., Yonekura-Sakakibara, K., Niida, R., Watanabe-Takahashi, A., & Saito, K. (2007). Phytochemical genomics in Arabidopsis thaliana: A case study for functional identification of flavonoid biosynthesis genes. Pure and Applied Chemistry, 79, 811–823.

    Article  CAS  Google Scholar 

  • Vallverdú-Queralt, A., Jáuregui, O., Medina-Remón, A., Andrés-Lacueva, C., & Lamuela-Raventós, R. M. (2010). Improved characterization of tomato polyphenols using liquid chromatography/electrospray ionization linear ion trap quadrupole Orbitrap mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Communications in Mass Spectrometry, 24, 2986–2992.

    Article  PubMed  Google Scholar 

  • Vukics, V., & Guttman, A. (2010). Structural characterization of flavonoid glycosides by multi-stage mass spectrometry. Mass Spectrometry Reviews, 29, 1–12.

    PubMed  CAS  Google Scholar 

  • Werner, E., Heilier, J. F., Ducruix, C., Ezan, E., Junot, C., & Tabet, J. C. (2008). Mass spectrometry for the identification of the discriminating signals from metabolomics: Current status and future trends. Journal of Chromatography B, 871, 143–163.

    Article  CAS  Google Scholar 

  • Wolfender, J. L., Waridel, P., Ndjoko, K., Hobby, K. R., Major, H. J., & Hostettmann, K. (2000). Evaluation of Q-TOF-MS/MS and multiple stage IT–MSn for the dereplication of flavonoids and related compounds in crude plant extracts. Analysis, 28, 895–906.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This research was granted by the Netherlands Metabolomics Centre (JJJvdH, JV, RCHdV) and the Centre for BioSystems Genomics (RJB, RCHdV), both of which are part of the Netherlands Genomics Initiative/Netherlands Organization for Scientific Research. The authors specifically thank Rik Kooke and Joost Keurentjes (Laboratory of Plant Physiology, WUR) for growing, harvesting and grinding the Arabidopsis ecotype population, Harry Jonker and Bert Schipper (PRI, WUR) for technical assistance, Piotr Kasper, Miguel Rojas, Thomas Hankemeier, and Rob Vreeken (LACDR, Leiden University) for valuable discussions on MSn fragmentation and Velitchka Mihaleva (Laboratory of Biochemistry, WUR) for fruitful comments on the manuscript.

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Authors and Affiliations

  1. Laboratory of Biochemistry, Wageningen University and Research Centre, Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands

    Justin J. J. van der Hooft & Jacques Vervoort

  2. Plant Research International, BU Bioscience, Wageningen University and Research Centre, P.O. Box 16, 6700 AA, Wageningen, The Netherlands

    Justin J. J. van der Hooft & Ric C. H. de Vos

  3. Laboratory of Plant Physiology, P.O. Box 658, 6700 AR, Wageningen, The Netherlands

    Raoul J. Bino

  4. Centre for BioSystems Genomics, P.O. Box 98, 6700 AB, Wageningen, The Netherlands

    Raoul J. Bino & Ric C. H. de Vos

  5. Netherlands Metabolomics Centre, Einsteinweg 55, 2333 CC, Leiden, The Netherlands

    Justin J. J. van der Hooft, Jacques Vervoort & Ric C. H. de Vos

Authors
  1. Justin J. J. van der Hooft
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  2. Jacques Vervoort
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  3. Raoul J. Bino
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  4. Ric C. H. de Vos
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Corresponding author

Correspondence to Justin J. J. van der Hooft.

Electronic supplementary material

Below is the link to the electronic supplementary material.

11306_2011_363_MOESM1_ESM.xls

The spectral data of ninety-nine tomato fruit metabolites and twenty-seven Arabidopsis thaliana metabolites are listed. (XLS 76 kb)

Supplementary material 2 (XLS 28 kb)

11306_2011_363_MOESM3_ESM.doc

The results of database searches for the elemental formulas of several tomato fruit metabolites are listed. (DOC 3696 kb)

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van der Hooft, J.J.J., Vervoort, J., Bino, R.J. et al. Spectral trees as a robust annotation tool in LC–MS based metabolomics. Metabolomics 8, 691–703 (2012). https://doi.org/10.1007/s11306-011-0363-7

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  • DOI: https://doi.org/10.1007/s11306-011-0363-7

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