Annotating unknown components from GC/EI-MS-based metabolite profiling experiments using GC/APCI(+)-QTOFMS

Abstract

GC/EI-MS-based metabolite profiling of derivatized polar fractions of crude plant extracts typically reveals several hundred components. Thereof, only up to one half can be identified using mass spectral and retention index libraries, the rest remains unknown. In the present work, the utility of GC/APCI(+)-QTOFMS for the annotation of unknown components was explored. Hence, EI and APCI(+) mass spectra of ~100 known components were extracted from GC/EI-QMS and GC/APCI(+)-QTOFMS profiles obtained from a methoximated and trimethylsilylated root extract of Arabidopsis thaliana. Based on this reference set, adduct and fragment ion formation under APCI(+) conditions was examined and the calculation of elemental compositions evaluated. During these studies, most of the components formed dominating protonated molecular ions. Despite the high mass accuracy (|Δm| ≤ 3 mDa) and isotopic pattern accuracy (mSigma ≤ 30) the determination of a component’s unique native elemental composition requires additional information, namely the number of trimethylsilyl and methoxime moieties as well as the analysis of corresponding collision-induced dissociation (CID) mass spectra. After all, the reference set was used to develop a strategy for the pairwise assignment of EI and APCI(+) mass spectra. Proceeding from these findings, the annotation of unidentified components detected by GC/EI-QMS using GC/APCI(+)-QTOFMS and corresponding deuterated derivatization reagents was attempted. For a total of 25 unknown components, pairs of EI and APCI(+) mass spectra were compiled and elemental compositions determined. Integrative interpretation of EI and CID mass spectra resulted in 14 structural hypotheses, of which seven were confirmed using authenticated standards.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. Amirav, A., Gordin, A., Poliak, M., & Fialkov, A. B. (2008). Gas chromatography–mass spectrometry with supersonic molecular beams. Journal of Mass Spectrometry, 43, 141–163.

    CAS  Article  PubMed  Google Scholar 

  2. Bellostas, N., Sorensen, A. D., Sorensen, J. C., & Sorensen, H. (2008). Fe2+-catalyzed formation of nitriles and thionamides from intact glucosinolates. Journal of Natural Products, 71, 76–80.

    CAS  Article  PubMed  Google Scholar 

  3. Birkemeyer, C., Luedemann, A., Wagner, C., Erban, A., & Kopka, J. (2005). Metabolome analysis: The potential of in vivo labeling with stable isotopes for metabolite profiling. Trends in Biotechnology, 23, 28–33.

    CAS  Article  PubMed  Google Scholar 

  4. Brenner, N., Haapala, M., Vuorensola, K., & Kostiainen, R. (2008). Simple coupling of gas chromatography to electrospray ionization mass spectrometry. Analytical Chemistry, 80, 8334–8339.

    CAS  Article  PubMed  Google Scholar 

  5. Bristow, T., Harrison, M., & Sims, M. (2010). The application of gas chromatography/atmospheric pressure chemical ionisation time-of-flight mass spectrometry to impurity identification in Pharmaceutical Development. Rapid Communications in Mass Spectrometry, 24, 1673–1681.

    CAS  Article  PubMed  Google Scholar 

  6. Carrasco-Pancorbo, A., Nevedomskaya, E., Arthen-Engeland, T., Zey, T., Zurek, G., Baessmann, C., et al. (2009). Gas chromatography/atmospheric pressure chemical ionization-time of flight mass spectrometry: Analytical validation and applicability to metabolic profiling. Analytical Chemistry, 81, 10071–10079.

    CAS  Article  PubMed  Google Scholar 

  7. Dunn, W., Erban, A., Weber, R. M., Creek, D., Brown, M., Breitling, R., et al. (2013). Mass appeal: metabolite identification in mass spectrometry-focused untargeted metabolomics. Metabolomics, 9, S44–S66.

    Article  Google Scholar 

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

    Article  PubMed  Google Scholar 

  9. Dunn, W. B., Broadhurst, D., Begley, P., Zelena, E., Francis-McIntyre, S., Anderson, N., et al. (2011). Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry. Nature Protocols, 6, 1060–1083.

    CAS  Article  PubMed  Google Scholar 

  10. Fiehn, O., Kopka, J., Dormann, P., Altmann, T., Trethewey, R. N., & Willmitzer, L. (2000a). Metabolite profiling for plant functional genomics. Nature Biotechnology, 18, 1157–1161.

    CAS  Article  PubMed  Google Scholar 

  11. Fiehn, O., Kopka, J., Trethewey, R. N., & Willmitzer, L. (2000b). Identification of uncommon plant metabolites based on calculation of elemental compositions using gas chromatography and quadrupole mass spectrometry. Analytical Chemistry, 72, 3573–3580.

    CAS  Article  PubMed  Google Scholar 

  12. Garcia-Villalba, R., Pacchiarotta, T., Carrasco-Pancorbo, A., Segura-Carretero, A., Fernandez-Gutierrez, A., Deelder, A. M., et al. (2011). Gas chromatography-atmospheric pressure chemical ionization-time of flight mass spectrometry for profiling of phenolic compounds in extra virgin olive oil. Journal of Chromatography A, 1218, 959–971.

    CAS  Article  PubMed  Google Scholar 

  13. Halket, J. M., & Zaikin, V. G. (2003). Derivatization in mass spectrometry-1. Silylation. European Journal of Mass Spectrometry, 9, 1–21.

    CAS  Article  PubMed  Google Scholar 

  14. Herebian, D., Hanisch, B., & Marner, F. J. (2005). Strategies for gathering structural information on unknown peaks in the GC/MS analysis of Corynebacterium glutamicum cell extracts. Metabolomics, 1, 317–324.

    CAS  Article  Google Scholar 

  15. Horning, E. C., Horning, M. G., Carroll, D. I., Dzidic, I., & Stillwell, R. N. (1973). New picogram detection system based on a mass spectrometer with an external ionization source at atmospheric pressure. Analytical Chemistry, 45, 936–943.

    CAS  Article  Google Scholar 

  16. Hummel, J., Selbig, J., Walther, D., & Kopka, J. (2007). The Golm Metabolome Database: A database for GC-MS based metabolite profiling. Topics in Current Genetics, 18, 75–95.

    CAS  Article  Google Scholar 

  17. Hummel, J., Strehmel, N., Selbig, J., Walther, D., & Kopka, J. (2010). Decision tree supported substructure prediction of metabolites from GC-MS profiles. Metabolomics, 6, 322–333.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Kind, T., Wohlgemuth, G., Lee, D. Y., Lu, Y., Palazoglu, M., Shahbaz, S., et al. (2009). FiehnLib: Mass spectral and retention index libraries for metabolomics based on quadrupole and time-of-flight gas chromatography/mass spectrometry. Analytical Chemistry, 81, 10038–10048.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Konishi, Y., Kiyota, T., Draghici, C., Gao, J. M., Yeboah, F., Acoca, S., et al. (2007). Molecular formula analysis by an MS/MS/MS technique to expedite dereplication of natural products. Analytical Chemistry, 79, 1187–1197.

    CAS  Article  PubMed  Google Scholar 

  20. Kopka, J. (2006). Current challenges and developments in GC-MS based metabolite profiling technology. Journal of Biotechnology, 124, 312–322.

    CAS  Article  PubMed  Google Scholar 

  21. Kopka, J., Schauer, N., Krueger, S., Birkemeyer, C., Usadel, B., Bergmuller, E., et al. (2005). GMD@CSB.DB: The Golm Metabolome Database. Bioinformatics, 21, 1635–1638.

    CAS  Article  PubMed  Google Scholar 

  22. Kumari, S., Stevens, D., Kind, T., Denkert, C., & Fiehn, O. (2011). Applying in-silico retention index and mass spectra matching for identification of unknown metabolites in accurate mass gc-tof mass spectrometry. Analytical Chemistry, 83, 5895–5902.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Lisec, J., Schauer, N., Kopka, J., Willmitzer, L., & Fernie, A. R. (2006). Gas chromatography mass spectrometry-based metabolite profiling in plants. Nature Protocols, 1, 387–396.

    CAS  Article  PubMed  Google Scholar 

  24. McEwen, C. N., & McKay, R. G. (2005). A combination atmospheric pressure LC/MS:GC/MS ion source: Advantages of dual AP-LC/MS:GC/MS instrumentation. Journal of the American Society of Mass Spectrometry, 16, 1730–1738.

    CAS  Article  Google Scholar 

  25. Pacchiarotta, T., Nevedomskaya, E., Carrasco-Pancorbo, A., Deelder, A. M., & Mayboroda, O. A. (2010). Evaluation of GC-APCI/MS and GC-FID as a complementary platform. Journal of Biomolecular Techniques, 21, 205–213.

    PubMed  PubMed Central  Google Scholar 

  26. Pfalz, M., Vogel, H., & Kroymann, J. (2009). The gene controlling the indole glucosinolate modifier1 quantitative trait locus alters indole glucosinolate structures and aphid resistance in Arabidopsis. Plant Cell, 21, 985–999.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. Portoles, T., Sancho, J. V., Hernandez, F., Newton, A., & Hancock, P. (2010). Potential of atmospheric pressure chemical ionization source in GC-QTOF MS for pesticide residue analysis. Journal of Mass Spectrometry, 45, 926–936.

    CAS  Article  PubMed  Google Scholar 

  28. Sanchez, D. H., Szymanski, J., Erban, A., Udvardi, M. K., & Kopka, J. (2010). Mining for robust transcriptional and metabolic responses to long-term salt stress: a case study on the model legume Lotus japonicus. Plant, Cell and Environment, 33, 468–480.

    CAS  Article  PubMed  Google Scholar 

  29. Schauer, N., Steinhauser, D., Strelkov, S., Schomburg, D., Allison, G., Moritz, T., et al. (2005). GC-MS libraries for the rapid identification of metabolites in complex biological samples. FEBS Letters, 579, 1332–1337.

    CAS  Article  PubMed  Google Scholar 

  30. Schiewek, R., Lorenz, M., Giese, R., Brockmann, K., Benter, T., Gab, S., et al. (2008). Development of a multipurpose ion source for LC-MS and GC-API MS. Analytical and Bioanalytical Chemistry, 392, 87–96.

    CAS  Article  PubMed  Google Scholar 

  31. Steinfath, M., Strehmel, N., Peters, R., Schauer, N., Groth, D., Hummel, J., et al. (2010). Discovering plant metabolic biomarkers for phenotype prediction using an untargeted approach. Plant Biotechnology Journal, 8, 900–911.

    CAS  Article  PubMed  Google Scholar 

  32. Strehmel, N., Hummel, J., Erban, A., Strassburg, K., & Kopka, J. (2008). Retention index thresholds for compound matching in GC-MS metabolite profiling. Journal of Chromatography B, 871, 182–190.

    CAS  Article  Google Scholar 

  33. von Wiren, N., Romheld, V., Shioiri, T., & Marschner, H. (1995). Competition between microorganisms and roots of barley and sorghum for iron accumulated in the root apoplasm. New Phytologist, 130, 511–521.

    Article  Google Scholar 

  34. Wachsmuth, C. J., Almstetter, M. F., Waldhier, M. C., Gruber, M. A., Nurnberger, N., Oefner, P. J., et al. (2011). Performance evaluation of gas chromatography-atmospheric pressure chemical ionization-time-of-flight mass spectrometry for metabolic fingerprinting and profiling. Analytical Chemistry, 83, 7514–7522.

    CAS  Article  PubMed  Google Scholar 

  35. Warren, C. (2013). Use of chemical ionization for GC–MS metabolite profiling. Metabolomics, 9, S110–S120.

    Article  Google Scholar 

  36. Zimmermann, R., Welthagen, W., & Groger, T. (2008). Photo-ionisation mass spectrometry as detection method for gas chromatography. Optical selectivity and multidimensional comprehensive separations. Journal of Chromatography A, 1184, 296–308.

    CAS  Article  PubMed  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to Christoph Böttcher.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Strehmel, N., Kopka, J., Scheel, D. et al. Annotating unknown components from GC/EI-MS-based metabolite profiling experiments using GC/APCI(+)-QTOFMS. Metabolomics 10, 324–336 (2014). https://doi.org/10.1007/s11306-013-0569-y

Download citation

Keywords

  • Atmospheric pressure chemical ionization
  • Electron ionization
  • Gas chromatography/mass spectrometry
  • Metabolite profiling
  • Quadrupole time-of-flight mass spectrometry
  • Structural elucidation