Simultaneous Measurement of Metabolite Concentration and Isotope Incorporation by Mass Spectrometry

Studies
of the topology, functioning, and regulation of metabolic
systems are based on two main types of information that can be measured
by mass spectrometry: the (absolute or relative) concentration of
metabolites and their isotope incorporation in 13C-labeling
experiments. These data are currently obtained from two independent
experiments because the 13C-labeled internal standard (IS)
used to determine the concentration of a given metabolite overlaps
the 13C-mass fractions from which its 13C-isotopologue
distribution (CID) is quantified. Here, we developed a generic method
with a dedicated processing workflow to obtain these two sets of information
simultaneously in a unique sample collected from a single cultivation,
thereby reducing by a factor of 2 both the number of cultivations
to perform and the number of samples to collect, prepare, and analyze.
The proposed approach is based on an IS labeled with other isotope(s)
that can be resolved from the 13C-mass fractions of interest.
As proof-of-principle, we analyzed amino acids using a doubly labeled 15N13C-cell extract as IS. Extensive evaluation
of the proposed approach shows a similar accuracy and precision compared
to state-of-the-art approaches. We demonstrate the value of this approach
by investigating the dynamic response of amino acids metabolism in
mammalian cells upon activation of the protein kinase R (PKR)-like
endoplasmic reticulum kinase (PERK), a key component of the unfolded
protein response. Integration of metabolite concentrations and isotopic
profiles reveals a reduced de novo biosynthesis of amino acids upon
PERK activation. The proposed approach is generic and can be applied
to other (micro)­organisms, analytical platforms, isotopic tracers,
or classes of metabolites.