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New mass spectrometric method for the separation of 13C and 15N-labelled amino acids

Isotope-based methods have been established for many years in biotechnological and medical research for the detailed investigation of cell metabolism. For the quantitative determination of intracellular metabolic fluxes, 13C-based metabolic flux analysis has become a routine method. In this model-based procedure, the relative fractions of enriched 13C isotopes in intracellular metabolites (so-called isotopologues) form the essential data basis. The latter can be measured using highly sensitive mass spectrometric methods.

 

The spectrum of measurable isotopologues has now been significantly extended by a novel analytical approach. This allows a simultaneous measurement of 13C- and 15N-isotopologues in amino acids. Thus, on the basis of a single labeling experiment, much more informative measurement data can be provided. The chemo-analytical basis is a previously overlooked property of amino acids with a primary amine during collision-induced dissociation in the mass spectrometer. Thus, the fraction of 13C- and 15N-isotopologues can be derived from specific fragment ions without having to separate them in the mass/charge domain. Although direct separation with more sensitive mass analyzers is possible, the measurement data obtained are less quantitative and therefore do not represent any added value for metabolic flux analysis.

 

In addition to the new analytical protocol, a ready-to-use data processing pipeline has been developed, which enables, among other things, the necessary isotope correction and direct integration of processed data into the correspondingly extended in-house software for metabolic flux analysis 13CFLUX2.

 

The study was published in the journal Analytical Chemistry and is accessible under:

 

https://pubs.acs.org/doi/10.1021/acs.analchem.9b01788

Graphic Neue massenspektrometrische Methode zur Trennung von 13C und 15N-markierten Aminosäuren


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