Fig. 6: Longitudinal dynamics and functional characterization of long-term proteomic panel biomarkers.
a Box plots with overlaid longitudinal trajectories illustrate the dynamic alterations in serological levels of long-term panel proteins (SERPINA7, CFHR4, CD163) between baseline (Week 0) and post-long-term AVT (Week 260) in fibrosis regressors versus non-regressors. Data are derived from the 4D-DIA-MS discovery cohort (training set: n = 15 pairs for R and n = 7 pairs for NR; testing set: n = 10 pairs for R and n = 4 pairs for NR) and the PRM-MS validation cohort (n = 42 pairs for R and n = 12 pairs for NR). All paired serum samples were biological replicates, with each pair originating from an individual patient. Normalized protein abundances were compared using a two-sided paired Student’s t-test (for normally distributed data) or a two-sided Wilcoxon signed-rank tests (for non-parametric data). A p-value < 0.05 was considered statistically significant. The box plots display the median (50th percentile; centre line), the 25th and 75th percentiles (box bounds), and the minima and maxima (whiskers) within 1.5 times the interquartile range (IQR) from the box bounds. b GSEA was performed on the complement and coagulation cascade pathway using proteins (CFHR4 and CD163) stratified into high- and low-expression groups based on median abundance thresholds in the 4D-DIA-MS discovery cohort. GSEA uses permutation-based significance testing to evaluate directional enrichment of gene sets in ranked gene lists, inherently employing a one-sided approach to detect coordinated over-representation at extremes of the expression profile. The enrichment plot displays the trajectory of the enrichment score across rank-ordered proteins, with the leading edge subset (indicated by the vertical red line) representing the proteins driving pathway enrichment. The p-value for the enrichment analysis is shown in the plot, highlighting the statistical significance of the enrichment. Abbreviations: 4D-DIA-MS four-dimensional data-independent acquisition mass spectrometry, NR non-regressor, PRM-MS parallel reaction monitoring mass spectrometry, R regressor.
