Fig. 3: Proteomics of metabolite extracts.
From: A diverse proteome is present and enzymatically active in metabolite extracts
a Schematic of sample preparation post-metabolite extraction for sample filtration, and bottom-up proteomics analysis. b BCA determination of protein content normalized to starting tissue amount. Significance calculated by Welch’s t-test (two-tailed, t-stat, df=2). Percentage protein content calculated as a fraction of the whole liver extract protein content across unfiltered (-) and filtered ( + F) AMW20 and AMW50 samples (mean ± SD, n = 3 technical replicates per group). c Peptide free protein content in AMW20 and AMW50 metabolite extractions. Data were log-transformed and analyzed using a linear regression model. Mean differences were calculated using the emmeans package in R. All mean differences are shown on the raw scale. Bar height represents the mean difference between AMW20 and AMW20F or AMW50 vs AMW50F from Fig. 3b. Error bars represents the upper 95% CI for the estimated mean difference. Data are normalized to the average total protein content in a whole liver (n = 3 technical replicates per group). d Number of proteins identified in AMW20, AMW50, and whole liver extracts (n = 3 technical replicates per group). e Heatmap depicting relative abundance of murine protein groups quantified across extraction conditions and row standardized. The top 50 most differentially abundant proteins (based on FDR-adjusted p-value) are shown. Significance calculated via LIMMA-eBayes (t-stat, two-sided) on Log2 transformed proteins without any missing values (n = 3 technical replicates per group). f PCA of AMW20, AMW30, and AMW50 protein groups in the murine liver (95% confidence ellipse, n = 3 technical replicates per group). g Number of proteins identified in AMW20, AMW35, and AMW50 metabolite extracts (n = 3 technical replicates per group). h Gene set enrichment analysis (biological processes) of proteins significantly impacted by water content. The top 4 pathways (based on FDR q-values) are labelled, and significantly enriched pathways relative to the whole liver metabolome by GeneRatio are colored in red.
