The natural stable isotope index for de novo structural elucidation of phytomolecules with geographical and biological tracing.

Not like the sequencing of genome and proteome that deals with the linear combination of base-pairs or amino acids, identification of unknown natural products has been always challenging due to diverse structures and various isomers. Searching fragment ions generated by mass spectrometers against databases has been the well-recognized approach. Because current searching algorithms generally compare unit masses and associated absolute intensities of ions, long lists of candidates with close similarity matching scores make it very difficult to achieve unambiguous structural identification. By using phytomolecules present in Allium sativum L. as examples, we demonstrated a natural stable isotope index (nSII) calculator for de novo structural elucidation along with geographical and biological tracing. The proposed nSII calculator provides a tool for mass spectrometric analysis of intramolecular- and compound-specific natural stable isotope ratios that are correlated with the activation or deactivation of metabolic pathways. It offers experimental evidences for the elucidation of elemental compositions, relationships among fragment ions and structural isoforms. Based on nSII calculator, a weighted mean index (wMI) has been established for the comprehensive examination of structural candidates, which inspects both mass spectrometric intensities and natural stable isotopes. Specific structural motifs that contain elements with specific isotopes are identified with unique nSII values, wMI values and accurate masses. Importantly, the nSII values are calculated as intensity ratios of heavy isotopes over light isotopes, which decreases interferences of signal fluctuations. Experimental results demonstrate that nSII and wMI values are useful to reject invalid candidates from the long lists of simple similarity matching reports, while other valid candidates may still be further confirmed with other techniques in addition to mass spectrometry. It should provide a label-free mass spectrometric approach for geographical and biological tracing of environment-metabolism interactions and enzyme-specific isotope effects if an isotopic ratio mass spectrometer (IR MS) can be combined.

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