Stable Isotope Labeling-Assisted Experiments

Stable isotope-based approaches are extensively utilized in untargeted metabolomics studies due to their significant potential for identifying unexpected and previously unknown metabolites.

Our core facility offers two types of isotope labeling-assisted experiments. Global metabolome labeling experiments enable the untargeted detection of hundreds to thousands of metabolites simultaneously, while studies using labeled tracers focus on investigating their biotransformation within biological systems.

Despite their differences, both approaches share the fundamental principle of generating characteristic isotope patterns. By analyzing samples with high-resolution mass spectrometry (HRMS) and processing the data using specialized software tools, it becomes possible to efficiently minimize noise, background signals, and any components lacking the isotope label.

We offer comprehensive and flexible support for your stable isotope labeling-assisted experiments, including assistance with experimental design, LC-HRMS sample analysis, automated data processing, and statistical evaluation of the results.

Global Metabolome Labeling Experiments

Stable isotope labeling of entire organisms can be achieved by continuously supplying labeled nutrients. For example, 13C labeling can be performed by feeding microorganisms with 13C-labeled carbon sources, such as glucose, in minimal media or by cultivating plants in a 13CO2​ atmosphere (e.g., using the Phytolabelbox). Similarly, other elements can also be labeled, such as nitrogen (15N), to specifically detect nitrogen-containing metabolites. This can be achieved by adding 15N-labeled compounds, such as labeled nitrogen sources, to the nutrient solution of growing plants.

When combined with native samples grown under identical conditions but provided with unlabeled nutrients, all metabolites of the organism are present in both native and isotope-labeled forms. Since elements like carbon and nitrogen are fundamental components of many metabolites, the global metabolome exhibits characteristic isotope patterns that can be detected using LC-HRMS methods. Conducting biological studies in the native system, combined with internal standardization using globally labeled counterparts, enables the investigation of global metabolome responses under specific experimental conditions.

For more details see:

  • Concept of stable isotope labelling studies: (Bueschl, Krska et al. 2013)
  • Workflow for global metabolome labelling experiments: (Bueschl, Kluger et al. 2014)
  • Software for automated data evaluation: (Bueschl, Kluger et al. 2017)
  • Study to investigate differences in the metabolome of diverse plant parts and comparison of extraction solvents on the metabolome: (Doppler, Kluger et al. 2016)
  • Production of 13C labelled plant material: (Ćeranić, Doppler et al. 2020)

Stable Isotope-Labeled Tracer Experiments

Stable isotope-labeled tracer experiments offer tremendous potential for truly untargeted evaluation of the (bio)transformation products derived from the applied tracer. Depending on the nature of the selected tracer, either only a labeled tracer (e.g., for endogenous metabolites such as phenylalanine) or a mixture of native and labeled forms of the tracer (e.g., for exogenous metabolites such as xenobiotics, including mycotoxins in plants) is introduced into the biological system under investigation.

During biotransformation, a characteristic isotope pattern is generated, which can be detected using LC-HRMS (e.g., QExactive Orbitrap HF). Automated data evaluation then allows for the efficient filtering and identification of all downstream metabolites derived from the tracer based on their specific isotope patterns.

For example applications see:

  • Mycotoxins in planta, food or mammalian cell cultures:  (Kluger, Bueschl et al. 2013, Kluger, Bueschl et al. 2015, Nathanail, Varga et al. 2015, Meng-Reiterer, Varga et al. 2017, Stadler, Lambertini et al. 2019, Flasch, Bueschl et al. 2020)
  • Phenylalanine in planta and cell cultures: (Kluger, Bueschl et al. 2014, Doppler, Bueschl et al. 2019, Doppler, Kluger et al. 2019)
  • Visualizing methanol derived artifacts: (Sauerschnig, Doppler et al. 2018)

Discover how stable isotope labeling can support your metabolomics research and help you achieve your scientific goals.

 

Contact us today to learn more about our services.

Related literature

Bueschl, C., B. Kluger, M. Lemmens, G. Adam, G. Wiesenberger, V. Maschietto, A. Marocco, J. Strauss, S. Bödi, G. Thallinger, R. Krska and R. Schuhmacher (2014). "A novel stable isotope labelling assisted workflow for improved untargeted LC–HRMS based metabolomics research." Metabolomics 10(4): 754-769.

Bueschl, C., B. Kluger, N. K. N. Neumann, M. Doppler, V. Maschietto, G. G. Thallinger, J. Meng-Reiterer, R. Krska and R. Schuhmacher (2017). "MetExtract II: A software suite for stable isotope assisted untargeted metabolomics." Analytical Chemistry 89(17): 9518-9526.

Bueschl, C., R. Krska, B. Kluger and R. Schuhmacher (2013). "Isotopic labeling-assisted metabolomics using LC–MS." Analytical and Bioanalytical Chemistry 405(1): 27-33.

Ćeranić, A., M. Doppler, C. Büschl, A. Parich, K. Xu, A. Koutnik, H. Bürstmayr, M. Lemmens and R. Schuhmacher (2020). "Preparation of uniformly labelled 13C- and 15N-plants using customised growth chambers." Plant Methods 16(1): 46.

Doppler, M., C. Bueschl, B. Kluger, A. Koutnik, M. Lemmens, H. Buerstmayr, J. Rechthaler, R. Krska, G. Adam and R. Schuhmacher (2019). "Stable Isotope–Assisted Plant Metabolomics: Combination of Global and Tracer-Based Labeling for Enhanced Untargeted Profiling and Compound Annotation." Frontiers in Plant Science 10(1366).

Doppler, M., B. Kluger, C. Bueschl, C. Schneider, R. Krska, S. Delcambre, K. Hiller, M. Lemmens and R. Schuhmacher (2016). "Stable Isotope-Assisted Evaluation of Different Extraction Solvents for Untargeted Metabolomics of Plants." International Journal of Molecular Sciences 17(7): 1017.

Doppler, M., B. Kluger, C. Bueschl, B. Steiner, H. Buerstmayr, M. Lemmens, R. Krska, G. Adam and R. Schuhmacher (2019). "Stable Isotope-Assisted Plant Metabolomics: Investigation of Phenylalanine-Related Metabolic Response in Wheat Upon Treatment With the Fusarium Virulence Factor Deoxynivalenol." Frontiers in Plant Science 10(1137).

Flasch, M., C. Bueschl, L. Woelflingseder, H. E. Schwartz-Zimmermann, G. Adam, R. Schuhmacher, D. Marko and B. Warth (2020). "Stable Isotope-Assisted Metabolomics for Deciphering Xenobiotic Metabolism in Mammalian Cell Culture." ACS Chemical Biology 15(4): 970-981.

Kluger, B., C. Bueschl, M. Lemmens, F. Berthiller, G. Häubl, G. Jaunecker, G. Adam, R. Krska and R. Schuhmacher (2013). "Stable isotopic labelling-assisted untargeted metabolic profiling reveals novel conjugates of the mycotoxin deoxynivalenol in wheat." Analytical and Bioanalytical Chemistry 405(15): 5031-5036.

Kluger, B., C. Bueschl, M. Lemmens, H. Michlmayr, A. Malachova, A. Koutnik, I. Maloku, F. Berthiller, G. Adam, R. Krska and R. Schuhmacher (2015). "Biotransformation of the mycotoxin deoxynivalenol in fusarium resistant and susceptible near isogenic wheat lines." PLoS ONE 10(3).

Kluger, B., C. Bueschl, N. Neumann, R. Stückler, M. Doppler, A. W. Chassy, A. L. Waterhouse, J. Rechthaler, N. Kampleitner, G. G. Thallinger, G. Adam, R. Krska and R. Schuhmacher (2014). "Untargeted profiling of tracer-derived metabolites using stable isotopic labeling and fast polarity-switching LC-ESI-HRMS." Analytical Chemistry 86(23): 11533-11537.

Meng-Reiterer, J., E. Varga, A. V. Nathanail, C. Bueschl, J. Rechthaler, S. P. McCormick, H. Michlmayr, A. Malachová, P. Fruhmann, G. Adam, F. Berthiller, M. Lemmens and R. Schuhmacher (2017). "Tracing the metabolism of HT-2 toxin and T-2 toxin in barley by isotope-assisted untargeted screening and quantitative LC-HRMS analysis." Analytical and Bioanalytical Chemistry 407(26).

Nathanail, A. V., E. Varga, J. Meng-Reiterer, C. Bueschl, H. Michlmayr, A. Malachova, P. Fruhmann, M. Jestoi, K. Peltonen, G. Adam, M. Lemmens, R. Schuhmacher and F. Berthiller (2015). "Metabolism of the Fusarium Mycotoxins T-2 Toxin and HT-2 Toxin in Wheat." Journal of Agricultural and Food Chemistry 63(35): 7862-7872.

Sauerschnig, C., M. Doppler, C. Bueschl and R. Schuhmacher (2018). "Methanol Generates Numerous Artifacts during Sample Extraction and Storage of Extracts in Metabolomics Research." Metabolites 8(1): 1.

Stadler, D., F. Lambertini, C. Bueschl, G. Wiesenberger, C. Hametner, H. Schwartz-Zimmermann, R. Hellinger, M. Sulyok, M. Lemmens, R. Schuhmacher, M. Suman, F. Berthiller and R. Krska (2019). "Untargeted LC–MS based 13C labelling provides a full mass balance of deoxynivalenol and its degradation products formed during baking of crackers, biscuits and bread." Food Chemistry 279: 303-311.