Comprehensive Metabolite Identification Strategy Using Multiple Two-Dimensional NMR Spectra of a Complex Mixture...

Identification of metabolites in complex mixtures represents a key step in metabolomics. A new strategy is introduced, which is implemented in a new public web server, COLMARm, that permits the coanalysis of up to three two-dimensional (2D) NMR spectra, namely, ¹³C-¹H HSQC (heteronuclear single quantum coherence spectroscopy), ¹H-¹H TOCSY (total correlation spectroscopy), and ¹³C-¹H HSQC-TOCSY, for the comprehensive, accurate, and efficient performance of this task. The highly versatile and interactive nature of COLMARm permits its application to a wide range of metabolomics samples independent of the magnetic field. Database query is performed using the HSQC spectrum, and the top metabolite hits are then validated against the TOCSY-type experiment(s) by superimposing the expected cross-peaks on the mixture spectrum. In this way the user can directly accept or reject candidate metabolites by taking advantage of the complementary spectral information offered by these experiments and their different sensitivities. The power of COLMARm is demonstrated for a human serum sample uncovering the existence of 14 metabolites that hitherto were not identified by NMR.