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. 2023 Jan 18;13(2):150. doi: 10.3390/metabo13020150

Figure 5.

Figure 5

Recovery and stability in E. coli metabolite extracts subjected to different extraction and sample purification procedures. Extract concentrations (mol/L) of selected intermediates of central carbon metabolism in (a) undiluted and (b) four-times diluted acetonitrile (ACN):water (1:1, v/v%) E. coli extracts cleared by size-exclusion centrifugation with a cutoff of 3 kDa. (c) Adenylate energy charges (AECs) in undiluted and four-times diluted E. coli extracts (left panel) and in E. coli extracts subjected to a solid phase extraction (SPE) lipid removal procedure involving size-exclusion centrifugation (right panel), all extracted in ACN:water (1:1, v/v%) and cleared by size-exclusion centrifugation with a cutoff of 3 kDa. (d) Extract concentrations (mol/L) of selected intermediates of central carbon metabolism and (e) AECs as measured in E. coli extracted in ACN:water (1:1, v/v%) and cleared by size-exclusion centrifugation with a cutoff of 3 or 10 kDa. For (a–e), the average and spread of n = 3–4 technical replicates are presented. (f) The fraction of propidium iodide (PI) and Syto9 stained cells in a non-extracted E. coli culture and an E. coli culture subjected to passive cold extraction (−20 °C, 30 min) in ACN:water:methanol (3:5:2, v/v%, 30 min), as estimated by fluorescence microscopy imagining. The average ± SD of n = 5 technical replicates is presented. > 200 cells were counted per technical replicate. (g) Extract concentrations (mol/L) of nucleotides and intermediates of glycolysis and the pentose phosphate pathway (PPP) in E. coli extracted by three repeated freeze-thaw cycles or by passive cold extraction (−20 °C, 30 min), both in ACN:water:methanol (3:5:2, v/v%). The average ± SD of n = 4 technical replicates is presented. (h) Extract concentrations (mol/L) of selected intermediates of central carbon metabolism and (i) AECs as measured in E. coli extracted in ACN:water (1:1, v/v%) or in ACN:water:methanol (3:5:2, v/v%). The average and spread of n = 4 technical replicates are presented. Significance levels from two-tailed T-tests assuming equal variances are marked **: p ≤ 0.01; ***: p ≤ 0.001, in (e,f,i). Metabolite abbreviations are listed in Supplementary Table S1.