Figure 3. Time course proteomics reveals complex dynamics of basement membrane assembly.

(A) Schematic of sample enrichment for matrix (ECM) proteins for tandem mass spectrometry (MS) analysis (created with BioRender.com). (B) Bar charts show the relative abundance of matrix and non-matrix proteins identified by MS analysis in the cellular and ECM fractions of kidney organoids at days 14, 18, and 25 (n = 3 pools per time point). Pooled data are presented as median, and error bars indicate the 95% confidence interval for the median. (C) Venn diagrams showi the identification overlap for matrix proteins detected in kidney organoids at days 14, 18, and 25. (D) Matrix proteins are classified as basement membrane, other structural and ECM-associated proteins. Bar charts show the number of matrix proteins per matrix category in both cellular and ECM fractions, and line charts show the changes in their relative abundance (percentage of total matrix abundance) over the time course differentiation. Pooled data are presented as median, and error bars indicate the 95% confidence interval for the median. (E) Protein interaction network showing all basement membrane proteins identified over the kidney organoid time course MS study (nodes represent proteins and connecting lines indicate reported protein-protein interactions). (F) Heat map showing the log₁₀-transformed abundance levels of basement membrane proteins identified in the ECM fraction along kidney organoid differentiation time course (proteins detected only at one time point are not shown). (G) Volcano plots show the log₂-fold change (x-axis) versus -log₁₀-p-value (y-axis) for proteins differentially expressed in the ECM fraction of kidney organoids from day 14 to day 18, and from day 18 to day 25 (n = 3 per time point). Key basement membrane proteins significantly upregulated (FC > 1.5, p-value<0.05, two-way ANOVA test, n = 3) are indicated. (H) Time-dependent changes in the relative abundance (percentage of total protein intensity) of key basement membrane proteins in the ECM fraction of kidney organoids during differentiation. Pooled data are presented as median. See Figure 3—figure supplement 1.

Figure 3—figure supplement 1. Time course proteomic analysis of kidney organoid differentiation.

(A) Gene Ontology (GO) term enrichment analysis of cellular component annotations associated with proteins detected in the cellular and extracellular matrix (ECM) fractions of kidney organoids by mass spectrometry (MS). GO terms were considered enriched when false discovery rate (FDR) < 0.10. (B) Protein interaction network depicts matrisome proteins identified by MS over kidney organoid differentiation time course (nodes represent proteins and connecting lines indicate reported protein-protein interactions). (C) Principal component analysis (PCA) for matrix proteins identified by MS in the cellular (left plot) and ECM (right plot) fractions of kidney organoids. (D) Top 15 most abundant basement membrane proteins found in kidney organoids. Proteins were ranked according to their normalized abundance levels (LFQ-intensities). Pooled data are shown as median, and error bars indicate the 95% confidence interval for the median. (E) Volcano plots show the log₂-fold change (x-axis) versus -log₁₀-p-value (y-axis) for proteins differentially expressed in the cellular fraction of kidney organoids from day 14 to day 18, and from day 18 to day 25. Key basement membrane proteins significantly upregulated (FC > 1.5, p-value<0.05, two-way ANOVA test, n = 3) are indicated. (F) Pathway enrichment analysis of proteins differentially expressed during kidney organoid differentiation: bar charts depict log-transformed FDR for the top-most enriched pathways (FDR < 0.10). Pathway terms shown were simplified.