Stable Isotope Assisted Assignment of Elemental Compositions for Metabolomics

Assignment of individual compound identities within
mixtures of thousands of metabolites in biological extracts
is a major challenge for metabolomic technology. Mass
spectrometry offers high sensitivity over a large dynamic
range of abundances and molecular weights but is limited
in its capacity to discriminate isobaric compounds. In this
article, we have extended earlier studies using isotopic
labeling for elemental composition elucidation (Rodgers,
R. P.; Blumer, E. N.; Hendrickson, C. L.; Marshall, A.
G. J. Am. Soc. Mass Spectrom. 2000, 11, 835−40) to
limit the formulas consistent with any exact mass measurement by comparing observations of metabolites extracted from Arabidopsis thaliana plants grown with (I)
12C and 14N (natural abundance), (II) 12C and 15N, (III)
13C and 14N, or (IV) 13C and 15N. Unique elemental
compositions were determined over a dramatically enhanced mass range by analyzing exact mass measurement
data from the four extracts using two methods. In the first,
metabolite masses were matched with a library of 11 000
compounds known to be present in living cells by using
values calculated for each of the four isotopic conditions.
In the second method, metabolite masses were searched
against masses calculated for a constrained subset of
possible atomic combinations in all four isotopic regimes.
In both methods, the lists of elemental compositions from
each labeling regime were compared to find common
formulas with similar retention properties by HPLC in at
least three of the four regimes. These results demonstrate
that metabolic labeling can be used to provide additional
constraints for higher confidence formula assignments
over an extended mass range.