High-Resolution Mass Spectrometry-Based Metabolomics for Increased Grape Juice Metabolite Coverage

Abstract

The composition of the juice from grape berries is at the basis of the definition of technological ripeness before harvest, historically evaluated from global sugar and acid contents. If many studies have contributed to the identification of other primary and secondary metabolites in whole berries, deepening knowledge about the chemical composition of the sole flesh of grape berries (i.e., without considering skins and seeds) at harvest is of primary interest when studying the enological potential of widespread grape varieties producing high-added-value wines. Here, we used non-targeted DI-FT-ICR-MS and RP-UHPLC-Q-ToF-MS analyses to explore the extent of metabolite coverage of up to 290 grape juices from four Vitis vinifera grape varieties, namely Chardonnay, Pinot noir, Meunier, and Aligoté, sampled at harvest from 91 vineyards in Europe and Argentina, over three successive vintages. SPE pretreatment of samples led to the identification of more than 4500 detected C,H,O,N,S-containing elemental compositions, likely associated with tens of thousands of distinct metabolites. We further revealed that a major part of this chemical diversity appears to be common to the different juices, as exemplified by Pinot noir and Chardonnay samples. However, it was possible to build significant models for the discrimination of Chardonnay from Pinot noir grape juices, and of Chardonnay from Aligoté grape juices, regardless of the geographical origin or the vintage. Therefore, this metabolomic approach opens access to a remarkable holistic molecular description of the instantaneous composition of such a biological matrix, which is the result of complex interplays among environmental, biochemical, and vine growing practices.

Keywords: Aligoté; Chardonnay; Meunier; Pinot noir; mesocarp; molecular fingerprints.

Figure 1

Display of the global grape juice metabolome detected by the different analytical strategies for SPE pretreated samples (top) and non SPE pretreated samples (bottom); (a1,b1) mass vs. O/C van Krevelen diagram representation of all detected DI-FT-ICR-MS mass peaks transformed into assigned elemental composition with an assignment error below 0.5 ppm, and a O/C ratio below 1.1. Dot sizes are proportional to the relative intensity of corresponding mass peaks, and with the following color codes: CHO (blue), CHON (orange), CHOS (green), CHONS (red); (a2,b2) all detected RP-UHPLC-Q-ToF-MS features represented as 2D maps projecting mass vs. RT (retention time).

Figure 2

Comparison of the global Chardonnay and Pinot noir metabolomes obtained by DI-FT-ICR-MS, considering all geographical regions and vintages. H/C vs. O/C van Krevelen diagram representation for Chardonnay (a) and Pinot noir (b) with the following color codes for elemental compositions: CHO (blue), CHON (orange), CHOS (green), CHONS (red). These diagrams represent mass peaks annotated into assigned elemental composition with an assignment error below 0.5 ppm, and an O/C ratio below 1.1, a H/C ratio below 1.5, and present in at least four samples for each variety. Dot sizes are proportional to the relative intensity of corresponding mass peaks. Comparison between annotated mass peaks for the two cultivars is reported in a Venn Diagram (c). Summary of comparisons of numbers of detected features between the two cultivars for the four analytical methods of this study (d).

Figure 3

Histogram (upper part) of the number of unambiguously assigned elemental compositions based on DI-FT-ICR-MS (column 1–2) and RP-UHPLC-Q-ToF-MS (column 3–4) detected m/z values with the following color codes for elemental compositions: CHO (blue), CHNO (orange), CHOS (green), CHNOS (red). Single dots below associate elemental compositions to the pretreatment used for the two MS analyses, and connected dots indicate that these compositions are common to two or more pretreatments and analyses, after peak alignment (column 5–9).

Figure 4

Multivariate statistical analyses of DI-FT-ICR-MS metabolomic analyses of SPE pretreated samples, regardless of the geographical origin or the vintage; supervised OPLS-DA discrimination of Chardonnay/Pinot noir grape juices (a) and Chardonnay/Aligoté grape juices (b); non supervised PCA analysis of Pinot noir and Meunier grape juices (c); each dot in (a–c) refers to a grape juice sample; jitter plots and boxplots showing examples of VIP m/z peaks for Chardonnay and Pinot noir (d) and for Chardonnay and Aligoté (e). Each jitter plot provides a density distribution of the corresponding mass peak intensity (expressed as the Log₁₀ value) among Chardonnay (yellow) and Pinot noir (purple) samples (d), and among Chardonnay (yellow) and Aligoté (green) samples (e); **, *** and **** indicate significance with p value at 1E−2, 1E−3, and 1E−4, respectively.