Stable isotope labelling assisted experiments

Stable isotope based concepts are widely used in untargeted metabolomics studies as they offer great potential to discover unexpected and unknown metabolites.

Two forms of isotope labelling assisted experiments are offered in the BMoSA core facility. Experiments with global metabolome labelling allow the untargeted detection of hundreds to thousands of metabolites in parallel while studies applying labelled tracers can be used to study its biotransformation in a biological system.

The core concept of generating characteristic isotope patterns however remains the same for both variants. Measuring the samples with high resolution MS and evaluation with a specific software tool (MetExtract II) allows efficient reduction of noise, background signals and everything that does not contain the isotope label.

We provide flexible services throughout your planned stable isotope labelling assisted experiments from assistance in design of experiment, LC-HRMS measurement of your samples, automated data evaluation to statistical analysis of the obtained results.

Stable isotope labelling of whole organisms can be achieved via continuous providing of labelled nutrients. ¹³C labeling, for example, can be achieved either by feeding with ¹³C labelled carbon sources like glucose for microorganism on minimal medium or via cultivating plants under ¹³CO₂ atmosphere (see Phytolabelbox). By combination with native samples, cultivated parallel under the same conditions but applying native nutrients, all metabolites of the respective organism are present in native and ¹³C labelled forms. Based on the omnipresence of carbon in all metabolites, the global metabolome represents the characteristic isotope pattern which can be detected with LC-HRMS methods. Performing biological studies in the native system and internal standardization with globally labelled counterparts allows to study the response of the global metabolome under the tested 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 labelled tracer experiments provide enormous potential for truly untargeted evaluation of (bio)transformation products of the applied tracer. Depending on the nature of the selected tracer either only a labelled tracer (for endogenous metabolites like phenylalanine) or a mix of native and labelled forms of the tracer (for exogenous metabolites like xenobiotics (e.g. mycotoxins in plants)) are applied on the biological system under investigation. A characteristic isotope pattern is formed during biotransformation. After analysis with LC-HRMS (see QExactive OrbitrapHF) automated data evaluation allows to efficiently filter all downstream metabolites formed out of the tracer via their specific isotope pattern.

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)

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.