Figure 4.

System-wide characterization of WP-induced abundance changes of Caco-2 proteins.

(A, B) Protein ratio variance between repeated LC/MS/MS analysis. (A) Profiles of protein ratio variance between repeated LC/MS/MS of 12 +/−WP sample pairs. (B) Scatter plot of protein ratio variance between repeated LC/MS/MS with respect to exponentially modified protein abundance index (emPAI) for an exemplary +/−WP sample pair. Proteins were ordered from high to low emPAI and assigned ranks where 1 corresponds to the highest emPAI. Color curves show the fraction of proteins with absolute FC variance (|ΔFC|) > 1.1 for each of the 12 sample pairs. FC, fold change. (C-D) Protein ratio variance with respect to the number of biological replicates. Ratios were from the average of two LC/MS/MS analysis. Ratio variances of +/−WP sample pair were compared between sample sizes of n+3 and n. Red, n = 3; blue, n = 6 and green, n = 9. s(n), sample size of n. (D) Scatter plots of protein ratio variance between s(n+3) and s(n) with respect to emPAI. Color curves show the fraction of proteins with |ΔFC| > 1.05. (E) Standard deviations of protein ratio at n = 9 biological replicates (“Std. Dev. (n=9)”, Supplemental Dataset 4 and 5) with respect to the number of significant peptide sequences for a linked protein; red dots and fitting line, BH p-value < 0.05; blue dots and fitting line, BH p-value ≥ 0.05; top marginal histogram, distribution of the number of unique peptide sequences; right marginal histogram, distribution of standard deviation. (F) Principal component analysis of the quantitative Caco-2 proteomes. Samples were grouped by ellipsoid according to treatment condition and duration. (G) Distribution of protein abundance changes induced by WP treatment over indicated periods. Proteins with positive or negative FCs are up- or down-regulated, respectively, under +WP conditions. Same proteins from the two different treatment durations are connected by solid lines with yellow ones highlighting significant abundance changes. The areas of dots are proportional to the standard deviations in protein abundance changes. The shade delimits −1.05 ≤ FC ≤ +1.05 or BH p-value ≤ 0.05. LTF, lactotransferrin. (H) Cross comparison of protein abundance change with respect to treatment duration. H1: mapping of 888 Caco-2 proteins (BH p < 0.05) from +/−WP (D9 → D15) dataset to +/−WP (D9 → D22) dataset. H2: mapping of 3,609 Caco-2 proteins (BH p < 0.05) from +/−WP (D9 → D22) dataset to +/−WP (D9 → D15) dataset. N = 12 for each group. To facilitate the visualization of small FCs, the color scale is limited to ±log2 1.0 with no distinction in colorimetric representation for greater magnitude of FC. In A and C: the vertical lines in box plots indicate the median and the whisker caps indicate the lower 10th and upper 90th percentiles, respectively.