UC Davis

Background: Metabolomic studies are targeted at identifying and

quantifying all metabolites in a given biological context. Among the tools used

for metabolomic research, mass spectrometry is one of the most powerful tools.

However, metabolomics by mass spectrometry always reveals a high number of

unknown compounds which complicate in depth mechanistic or biochemical

understanding. In principle, mass spectrometry can be utilized within

strategies of de novo structure elucidation of small molecules, starting with

the computation of the elemental composition of an unknown metabolite using

accurate masses with errors < 5 ppm (parts per million). However even with

very high mass accuracy (< 1 ppm) many chemically possible formulae are

obtained in higher mass regions. In automatic routines an additional orthogonal

filter therefore needs to be applied in order to reduce the number of potential

elemental compositions. This report demonstrates the necessity of isotope

abundance information by mathematical confirmation of the concept. Results:

High mass accuracy (< 1 ppm) alone is not enough to exclude enough

candidates with complex elemental compositions (C, H, N, S, O, P, and

potentially F, Cl, Br and Si). Use of isotopic abundance patterns as a single

further constraint removes > 95% of false candidates. This orthogonal filter

can condense several thousand candidates down to only a small number of

molecular formulas. Example calculations for 10, 5, 3, 1 and 0.1 ppm mass

accuracy are given. Corresponding software scripts can be downloaded from

http:// fiehnlab. ucdavis. edu. A comparison of eight chemical databases

revealed that PubChem and the Dictionary of Natural Products can be recommended

for automatic queries using molecular formulae. Conclusion: More than 1.6

million molecular formulae in the range 0 - 500 Da were generated in an

exhaustive manner under strict observation of mathematical and chemical rules.

Assuming that ion species are fully resolved (either by chromatography or by

high resolution mass spectrometry), we conclude that a mass spectrometer

capable of 3 ppm mass accuracy and 2% error for isotopic abundance patterns

outperforms mass spectrometers with less than 1 ppm mass accuracy or even

hypothetical mass spectrometers with 0.1 ppm mass accuracy that do not include

isotope information in the calculation of molecular formulae.