Fig. 6: Untargeted metabolomics reveals time-dependent, protein mediated effects in dried and resuspended metabolomics extracts.
From: A diverse proteome is present and enzymatically active in metabolite extracts
a Upset plot showing intersections of features with time dependent drift as defined by significantly non-zero slopes using a generalized additive model. Bars corresponding to individual extraction conditions are colored: AMW20 (light blue, filled), AMW20 + F (light blue, open), AMW50 (dark blue, filled), and AMW50 + F (dark blue, open). b PCA of murine liver metabolites from AMW20 ±Filter and AMW50 ±Filter murine liver samples over time (25 repeat injections, 0-84 hr post-resuspension). c PCA scores plot of liver metabolites from AMW50 samples showing injection order-dependent metabolite drift (25 repeat injections, 0-84 hr post-resuspension). d PCA loadings of murine liver metabolites from AMW50 samples over time (25 repeat injections 0–84 hr post-resuspension). Red dots represent the most positive and negative values along PC1 ( + 0.073, m/z 279.1137 at RT 12.066; no formula prediction and −0.073; m/z 320.0924 at RT 8.792; predicted chemical formula C11H19N3O6S Δppm = 1.0). e MS1 mass spectra at RT = 12.066 showing γ-glutamyl-glutamate (Glu-Glu) in murine liver following D5-glutamate addition at resuspension. Representative AMW50 sample spectrum is shown. Peaks corresponding to unlabeled Glu-Glu (275.0887 m/z), D4-Glu-Glu (279.1137 m/z), and D5-Glu-Glu (280.1199 m/z) are shown. f Relative abundance of D5-glutamate labeled glutamyl-glutamate (Glu-Glu) over time (25 repeat injections 0-84 hr post-resuspension) in AMW50 (solid) samples. g Relative abundance of D5-glutamate-labeled glutamate over time (25 repeat injections 0-84 hr post-resuspension) in AMW50 (solid) and AMW50 + F (dashed) samples. h Relative abundance of GSH, GSSH, and Me-GSH over time (25 repeat injections 0-84 hr post-resuspension) in AMW50 (solid) and AMW50 + F (dashed) samples. i Relative abundance of murine GSR and GSTs in metabolite extract across AMW20, AMW35, and AMW50 extraction conditions. Main effect FDR value shown (mean ± SD, n = 3 technical replicates per group). j Schematic showing GSR-mediated glutathione reduction, spontaneous glutathione oxidation, and GST-mediated glutathionylation. k Precursor ions with glutathione fragment ion, m/z = 308.0912 using the T3 method. Number of chromatographic peaks identified with a peak width greater than 6 s, and peak intensity 1x105 or greater. Representative chromatogram for precursor ion m/z 464.2061 (± 5ppm), and the ddMS2 for that ion at RT = 9.0 min.
