Can we trust untargeted metabolomics? Results of the metabo-ring initiative, a large-scale, multi-instrument inter-laboratory study.

The metabo-ring initiative brought together five nuclear magnetic resonance instruments (NMR) and 11 different mass spectrometers with the objective of assessing the reliability of untargeted metabolomics approaches in obtaining comparable metabolomics profiles. This was estimated by measuring the proportion of common spectral information extracted from the different LCMS and NMR platforms. Biological samples obtained from 2 different conditions were analysed by the partners using their own in-house protocols. Test #1 examined urine samples from adult volunteers either spiked or not spiked with 32 metabolite standards. Test #2 involved a low biological contrast situation comparing the plasma of rats fed a diet either supplemented or not with vitamin D. The spectral information from each instrument was assembled into separate statistical blocks. Correlations between blocks (e.g., instruments) were examined (RV coefficients) along with the structure of the common spectral information (common components and specific weights analysis). In addition, in Test #1, an outlier individual was blindly introduced, and its identification by the various platforms was evaluated. Despite large differences in the number of spectral features produced after post-processing and the heterogeneity of the analytical conditions and the data treatment, the spectral information both within (NMR and LCMS) and across methods (NMR vs. LCMS) was highly convergent (from 64 to 91 % on average). No effect of the LCMS instrumentation (TOF, QTOF, LTQ-Orbitrap) was noted. The outlier individual was best detected and characterised by LCMS instruments. In conclusion, untargeted metabolomics analyses report consistent information within and across instruments of various technologies, even without prior standardisation.

A High-Throughput UHPLC-QqQ-MS Method for Polyphenol Profiling in Rosé Wines.

A rapid, sensitive and selective analysis method using Ultra High Performance Liquid Chromatography coupled to triple-quadrupole Mass Spectrometry (UHPLC-QqQ-MS) has been developed for the quantification of polyphenols in rosé wines. The compound detection being based on specific MS transitions in Multiple Reaction Monitoring (MRM) mode, the present method allows the selective quantification of up to 152 phenolic and two additional non-phenolic wine compounds in 30 min without sample purification or pre-concentration, even at low concentration levels. This method was repeatably applied to a set of 12 rosé wines and thus proved to be suitable for high-throughput and large-scale metabolomics studies.

How to gain insight into the polydispersity of tannins: a combined MS and LC study.

In the context of the potential health benefits of food polyphenols, the bioavailability of tannins (i.e. proanthocyanidins) is a major issue, which is strongly influenced by the polydispersity and the degree of polymerisation of tannins. The average degree of polymerisation (DP) of tannins is usually determined using depolymerisation methods, which do not provide any information about their polymer distribution. Moreover, it is still a challenge to characterise tannin fractions of high polydispersity and/or containing polymers of high molecular weights, due to the limit of detection of direct mass spectrometry (MS) analysis methods. In the present work, the polydispersity of several tannin fractions is investigated by two complementary methods: a MALDI-MS method and a semi-preparative sub-fractionation. Using a combination of these methods we are able to gain insight into the DP distributions of the fractions consisting of tannins of medium and high DP. Moreover combining analyses can be useful to assess and compare the DP distributions of most tannin fractions.

Effect of enzyme additions on the oligosaccharide composition of Monastrell red wines from four different wine-growing origins in Spain.

The release of oligosaccharides during winemaking depends on the grape skin cell wall degradation, which can be facilitated by the use of enzymes. Oligosaccharide quantities and composition in wine could be influenced by the "terroir" effect. Monastrell wine was elaborated from grapes from four different "terroirs" (Cañada Judío, Albatana, Chaparral-Bullas and Montealegre). Monastrell wines were also treated with ß-galactosidase enzyme addition and commercial enzyme addition. The results showed significant differences in the Monastrell wine oligosaccharide fractions, according to the geographical origin of grapes. A higher quantity of oligosaccharides was found for three out of four terroirs studied when commercial enzymes were added. The use of commercial enzyme modified the Arabinose/Galactose and the Rhamnose/Galacturonic acid ratios in Cañada Judío and Albatana terroirs wines, and it modified the (Arabinose+Galactose)/Rhamnose ratio in Cañada Judío, Albatana and Chaparral-Bullas terroirs wines. Therefore, the "terroir" impacts the effect of commercial enzyme treatment on wine oligosaccharide composition.

A steroidomic approach for biomarkers discovery in doping control.

Anti-doping authorities have high expectations of the athlete steroidal passport (ASP) for anabolic-androgenic steroids misuse detection. However, it is still limited to the monitoring of known well-established compounds and might greatly benefit from the discovery of new relevant biomarkers candidates. In this context, steroidomics opens the way to the untargeted simultaneous evaluation of a high number of compounds. Analytical platforms associating the performance of ultra-high pressure liquid chromatography (UHPLC) and the high mass-resolving power of quadrupole time-of-flight (QTOF) mass spectrometers are particularly adapted for such purpose. An untargeted steroidomic approach was proposed to analyse urine samples from a clinical trial for the discovery of relevant biomarkers of testosterone undecanoate oral intake. Automatic peak detection was performed and a filter of reference steroid metabolites mass-to-charge ratio (m/z) values was applied to the raw data to ensure the selection of a subset of steroid-related features. Chemometric tools were applied for the filtering and the analysis of UHPLC-QTOF-MS(E) data. Time kinetics could be assessed with N-way projections to latent structures discriminant analysis (N-PLS-DA) and a detection window was confirmed. Orthogonal projections to latent structures discriminant analysis (O-PLS-DA) classification models were evaluated in a second step to assess the predictive power of both known metabolites and unknown compounds. A shared and unique structure plot (SUS-plot) analysis was performed to select the most promising unknown candidates and receiver operating characteristic (ROC) curves were computed to assess specificity criteria applied in routine doping control. This approach underlined the pertinence to monitor both glucuronide and sulphate steroid conjugates and include them in the athletes passport, while promising biomarkers were also highlighted.

Effect of macerating enzymes on the oligosaccharide profiles of Merlot red wines.

Commercial pectinase preparations are applied in winemaking to improve wine processing and final quality. These preparations contain pectolytic enzyme activities such as polygalacturonases, pectin esterases, pectin lyases, and rhamnogalacturonases. These enzymes modify the polysaccharide and oligosaccharide composition of wines. The influence of various commercial enzyme preparations on wine oligosaccharide composition was studied, on Merlot wines from the Bordeaux area. Wine oligosaccharides were isolated by high-resolution size-exclusion chromatography on a Superdex-30 HR column. The glycosyl residue and glycosyl linkage compositions of the oligosaccharide fractions obtained were determined. The MS spectra of the Merlot oligosaccharide fractions from control and enzyme-treated wines were recorded on an AccuTOF mass spectrometer equipped with an electrospray ionization (ESI) source and a time-of-flight (TOF) mass analyzer. Oligosaccharides in the control wines were partly methylated homogalacturonans, corresponding to smooth regions of pectins, whereas those of the enzyme-treated wines were mostly rhamnogalacturonan-like structures linked with neutral lateral chains, arising from the hairy regions. The enzyme preparations used thus cleaved the rhamnogalacturonan backbone of the hairy zones and demethylated and hydrolyzed the smooth regions. Besides, different structures were detected, depending on the enzyme preparation used, indicating that they contained rhamnogalacturonase activities with different specificities. The oligosaccharide profiles can serve as a marker of enzymatic treatments.

Comprehensive study of condensed tannins by ESI mass spectrometry: average degree of polymerisation and polymer distribution determination from mass spectra.

The determination of the molecular mass distribution of tannins is still a challenge. To elucidate it, mass spectrometry is potentially interesting, but many previous studies have highlighted that the mass spectra of a tannin fraction do not always reflect the actual abundance of different chain lengths. To clarify the potentialities offered by the MS approach, a comprehensive study involving different tannin fractions analysed under different conditions was conducted with an electrospray ionization (ESI) source. This study allowed optimised ESI-MS conditions to be established for analysing tannins but also it outlines the limits of detection encountered. If the detection of high molecular weight tannins seems difficult or even impossible, the spectral distortions brought about by this limitation are not totally related to the sole average degree of polymerisation of the tannin fraction studied but greatly depend on its polymer distribution. However, ESI-MS used under optimised conditions is a suitable method to study tannin composition of vegetable extracts which contain degree of polymerisations below 26.

Influence of the glycosylation of human salivary proline-rich proteins on their interactions with condensed tannins.

Binding of condensed tannins to salivary proteins is supposed to be involved in their astringency. First, complexes arising from the interaction of saliva from two individuals and tannins were studied. Then interaction mixture models containing purified saliva proteins were developed. The highest polymerized tannins predominantly precipitated together with the salivary proteins. Electrophoresis of proteins in combination with thiolysis analysis of tannins indicated proline-rich protein (PRP)-polyphenol complexes in precipitated fractions and also in the soluble ones with individual differences. Individual salivas exhibiting different protein patterns were discriminated with regard to their ability to interact with tannins. From binding studies with purified classes of salivary proteins, interactions were shown to depend on the nature of the protein, in particular on their glycosylation state. For low concentrations of tannins, glycosylated PRP-tannin interactions led to complexes that remained soluble, whereas those arising from nonglycosylated PRP-tannin interactions were precipitated. This finding could indicate that under physiological conditions, complexes involving glycosylated proteins maintain part of the lubrication of the oral cavity, whereas tannin trapping leads to a lower astringency perception.

Direct mass spectrometry approaches to characterize polyphenol composition of complex samples.

Lower molecular weight polyphenols including proanthocyanidin oligomers can be analyzed after HPLC separation on either reversed-phase or normal phase columns. However, these techniques are time consuming and can have poor resolution as polymer chain length and structural diversity increase. The detection of higher molecular weight compounds, as well as the determination of molecular weight distributions, remain major challenges in polyphenol analysis. Approaches based on direct mass spectrometry (MS) analysis that are proposed to help overcome these problems are reviewed. Thus, direct flow injection electrospray ionization mass spectrometry analysis can be used to establish polyphenol fingerprints of complex extracts such as in wine. This technique enabled discrimination of samples on the basis of their phenolic (i.e. anthocyanin, phenolic acid and flavan-3-ol) compositions, but larger oligomers and polymers were poorly detectable. Detection of higher molecular weight proanthocyanidins was also restricted with matrix-assisted laser desorption ionization (MALDI) MS, suggesting that they are difficult to desorb as gas-phase ions. The mass distribution of polymeric fractions could, however, be determined by analyzing the mass distributions of bovine serum albumin/proanthocyanidin complexes using MALDI-TOF-MS.