2 research outputs found
Isotopic Studies of Metabolic Systems by Mass Spectrometry: Using Pascalâs Triangle To Produce Biological Standards with Fully Controlled Labeling Patterns
Mass spectrometry (MS) is widely
used for isotopic studies of metabolism
in which detailed information about biochemical processes is obtained
from the analysis of isotope incorporation into metabolites. The biological
value of such experiments is dependent on the accuracy of the isotopic
measurements. Using MS, isotopologue distributions are measured from
the quantitative analysis of isotopic clusters. These measurements
are prone to various biases, which can occur during the experimental
workflow and/or MS analysis. The lack of relevant standards limits
investigations of the quality of the measured isotopologue distributions.
To meet that need, we developed a complete theoretical and experimental
framework for the biological production of metabolites with fully
controlled and predictable labeling patterns. This strategy is valid
for different isotopes and different types of metabolisms and organisms,
and was applied to two model microorganisms, Pichia
augusta and Escherichia coli, cultivated on <sup>13</sup>C-labeled methanol and acetate as sole
carbon source, respectively. The isotopic composition of the substrates
was designed to obtain samples in which the isotopologue distribution
of all the metabolites should give the binomial coefficients found
in Pascalâs triangle. The strategy was validated on a liquid
chromatographyâtandem mass spectrometry (LC-MS/MS) platform
by quantifying the complete isotopologue distributions of different
intracellular metabolites, which were in close agreement with predictions.
This strategy can be used to evaluate entire experimental workflows
(from sampling to data processing) or different analytical platforms
in the context of isotope labeling experiments
Improved Isotopic Profiling by Pure Shift Heteronuclear 2D <i>J</i>âResolved NMR Spectroscopy
Quantitative information
on the carbon isotope content of metabolites
is essential for flux analysis. Whereas this information is in principle
present in proton NMR spectra through both direct and long-range heteronuclear
coupling constants, spectral overlap and homonuclear coupling constants
both hinder its extraction. We demonstrate here how pure shift 2D <i>J</i>-resolved NMR spectroscopy can simultaneously remove the
homonuclear couplings and separate the chemical shift information
from the heteronuclear coupling patterns. We demonstrate the power
of this method on cell lysates from different bacterial cultures and
investigate in detail the branched chain amino acid biosynthesis