Small Molecule Isotope Resolved Formula Enumeration: A Methodology for Assigning Isotopologues and Metabolite Formulas in Fourier Transform Mass Spectra

Improvements in Fourier
transform mass spectrometry (FT-MS) enable
increasingly more complex experiments in the field of metabolomics.
What is directly detected in FT-MS spectra are spectral features (peaks)
that correspond to sets of adducted and charged forms of specific
molecules in the sample. The robust assignment of these features is
an essential step for MS-based metabolomics experiments, but the sheer
complexity of what is detected and a variety of analytically introduced
variance, errors, and artifacts has hindered the systematic analysis
of complex patterns of observed peaks with respect to isotope content.
We have developed a method called SMIRFE that detects small biomolecules
and determines their elemental molecular formula (EMF) using detected
sets of isotopologue peaks sharing the same EMF. SMIRFE does not use
a database of known metabolite formulas; instead a nearly comprehensive
search space of all isotopologues within a mass range is constructed
and used for assignment. This search space can be tailored for different
isotope labeling patterns expected in different stable isotope tracing
experiments. Using consumer-level computing equipment, a large search
space of 2000 Da was constructed, and assignment performance was evaluated
and validated using verified assignments on a pair of peak lists derived
from spectra containing unlabeled and 15N-labeled versions
of amino acids derivatized using ethylchloroformate. SMIRFE identified
18 of 18 predicted derivatized EMFs, and each assignment was evaluated
statistically and assigned an e-value representing the probability
to occur by chance.