Determination of optimal initial sample weight and extraction solvents

Before the time-course experiment was performed, the seven days old VPN cell cultures were analysed to investigate the minimal weight needed for targeted metabolomics analysis. This parameter is of great importance to minimize the effects of the sampling on the cell culture dynamics. In this preliminary experiment, 100 mg and 300 mg of fresh frozen samples and 30 mg and 50 mg of freeze-dried samples were used with two extraction solvent mixtures. The results indicated that the concentration of total polyphenols were not different among fresh frozen and freeze dried samples by extraction with cold methanol. Nonetheless, a significantly higher total polyphenol concentration was found in freeze-dried samples extracted by chloroform/methanol/water mixture. Total polyphenols in the fresh frozen samples were not different between the two extraction methods, regardless of the initial sample weight (Fig 8). It was decided that 100 mg fresh frozen samples could be the minimal initial weight of cell cultures to be able to extract metabolites and allow multiple sample collections from the same culture flask.

Figure 8 Total polyphenol concentrations in VPN cell cultures with different initial sample weight and extraction methods

F100C- 100 mg fresh frozen cells extracted by chloroform/methanol/water F100M- 100 mg fresh frozen cells extracted by cold absolute methanol F300C- 300 mg fresh frozen cells extracted by chloroform/methanol/water F300M- 300 mg fresh frozen cells extracted by cold absolute methanol FFD30C- 30 mg freeze-dried cells extracted by chloroform/methanol/water FFD30M- 30 mg freeze-dried cells extracted by cold absolute methanol FFD50C- 50 mg freeze-dried cells extracted by chloroform/methanol/water FFD50M- 50 mg freeze-dried cells extracted by cold absolute methanol

To evaluate the effects of the two extraction methods, 100 mg of fresh frozen cells of VPN were collected at 12 time points (between day 0 to day 14). Each sample consisted of three biological replicates. The UPLC-MS/MS based targeted metabolomics analysis was already proven suitable to analyse polyphenols in grape berries (Vrhovsek et al. 2012). Using this optimized protocol of chromatography and mass spectrometric detection, 16 (poly)phenolic compounds in VPN cell suspension cultures could be identified. To obtain the first overview of the analysis of all detected phenolic acids, stilbenes, flavonols and flavan-3-ols in the grape cells, heat maps are created for all 16 UPLC-MS/MS metabolite profiles from 12 time points (Fig 9). The overall profiles of polyphenols appeared to be higher in the ternary mixture of solvent chloroform/methanol/water than in absolute methanol. In terms of absolute concentrations, the concentration of total phenols during the 15 days of growing period ranged between 0.13 mg/g and 3.95 mg/g with the mixture containing chloroform. In the same period, the extraction with absolute methanol yielded only 0.06-0.87 mg/g.

Figure 9 Heatmap visualising the concentrations (in logarithmic scale) of the metabolites in the VPN cell suspension cultures by two extraction methods.

To get an immediate overview of the patterns in the data, and to see whether these patterns are associated with extraction methods, PCA was performed. PCA biplot (Fig 10) revealed obvious clustering patterns of two extraction methods for grape cells, suggesting good analytical resolution that can distinguish different extracted metabolic profiles in the cells. The variables related to these two extraction methods are shown as loadings in the biplot. The samples extracted with chloroform method appears to be separated due to the high concentrations of isorhapontin, flavonoids such as quercetin 3-O-glucuronide, phenylpropanoid derivatives such as caftaric acid as well as vanillin.

Figure 10 PCA biplot shows the projection of the samples from two extraction solvents in the PC1 and PC2 plane. The loadings show the contribution of the different metabolites. The same color shows the extraction methods.

A more clear view of the yield of phenolic compounds determined by UPLC-MS/MS in the extracts is presented in Fig 11. The graphical presentation clearly indicates the differences between two solvents used, which is also supported by a statistical analysis (two samples t-test at p < 0.05). Chloroform/methanol/water mixture gave much higher yields of phenolic compounds than pure methanol, and this difference can mainly be attributed to the higher quantities of stilbene compounds for all time points.

Figure 11 Total content (in square-root scale) of polyphenols in VPN cell cultures (expressed as mg/g fresh cells) in the extracts of chloroform/methanol/water and methanol.

Aiming to evaluate the repeatability of the sample preparation procedure and measurements, the relative standard deviation (RSD) of the concentration of metabolites were measured. These values are determined using the same biological samples for the two extraction methods. The comparison of the distribution of the RSD between the extraction mixtures is presented as histograms in Fig 12. The compounds extracted by methanol were detected in lower and varying amounts; most of them are lower than the limit of quantification (LOQ). The majority of the metabolites extracted by methanol displayed a RSD of the area between 50-100%. The metabolites extracted with chloroform/methanol/water mixture have lower RSD than those extracted with methanol, suggesting that samples with chloroform/methanol/water mixture gave the more precise metabolite abundance.

Figure 12 Histograms of relative standard deviations (RSDs) of metabolite abundancy for two extraction methods.

2.3.2. Comparative analysis of secondary metabolites in V. vinifera cv. Pinot Noir