Comprehensive Metabolite Identification Strategy Using Multiple Two-Dimensional NMR Spectra of a Complex Mixture Implemented in the COLMARm Web Server

Abstract

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 co-analysis of up to three 2D NMR spectra, namely 13C-1H HSQC, 1H-1H TOCSY, 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.

Coadministration of the Campylobacter jejuni N-Glycan-Based Vaccine with Probiotics Improves Vaccine Performance in Broiler Chickens

“…Furthermore, the data were scaled by using nonthreshold logarithmic transformation in order to carry out multivariate analyses, PCA and PLS-DA, to identify sources of variation that could separate the data across the different study groups. Putative metabolite identification was carried out by using the database-matching platforms COLMAR (87) and Assure NMR (BrukerBiospin). …”

Non‐Uniform and Absolute Minimal Sampling for High‐Throughput Multidimensional NMR Applications

“…In the case of AMS TOCSY, the user can also upload the peak list (together with peak integrals) so that the server can directly use the peak list for analysis and use the uploaded spectrum for visualization only. As an additional option, the web server also provides spectral referencing based on pattern matching of commonly found metabolites …”

“…[55][56][57][58][59][60][61] For the automated analysis of multi-dimensional NMRs pectra of metabolomics samples, available softwarea nd webservers are more limited. [62,63] For 2D TOCSY (or 3D HSQC-TOCSY experiments) of complex mixtures, the large number of cross-peaks makes the manual analysiso fs uch spectra af ormidable challenge. Af irst step in spectrala nalysis is the identification of cross-peaks and their positionst hrough peak-picking.…”

(3, 2)D 1H, 13C BIRDr,X-HSQC-TOCSY for NMR structure elucidation of mixtures: application to complex carbohydrates

“…This separation is crucial for establishing networks of coupled spins by hyphenated techniques that combine homo-and heteronuclear polarisation transfers, such as 2D 1 H-13 C HSQC-TOCSY. Indeed, 2D 1 H-13 C HSQC-TOCSY spectra have been used to analyse complex oligosaccharides (Rodriguez-Carvajal et al 2003;Sato et al 2008;Panagos et al 2012), polysaccharides (Uhrin et al 1994), glycoproteins (Debeer et al 1994), peptides (Sonti et al 2012), fatty acids (Willker and Leibfritz 1998), metabolites (Bingol and Bruschweiler 2011;Bingol et al 2014Bingol et al , 2016, or food material (Kew et al 2017;Wei et al 2011). However, as the sample complexity increases, 13 C chemical shifts stop being unique at some point, making the interpretation of 2D HSQC-TOCSY spectra problematic.…”