Longitudinal stability of urinary extracellular vesicle protein patterns within and between individuals

Figures

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  Figure 1. Schematic workflow of the study and overview of the whole urinary EV proteome. (a) Schematic diagram of the collection of urine samples on the upper panel, and DIA-MS workflow on the lower panel. Urine was collected from 8 individuals at 9 timepoints over the course of 6 months (total 72 samples) and was pre-cleared from dead and apoptotic cells using centrifugation and stored at − 80 °C. 50 ml urine per donor was concentrated to 1 ml with ultrafiltration (100-kDa cut-off) and urinary EVs were subsequently isolated using the Vn96-peptide-affinity kit20. For the high-depth spectral library generation, gender-specific urinary EV pools (n = 2) were subjected to 10-band gel fractionation followed by DDA-MS. Individual samples (n = 72) were measured in single shot DIA-MS mode, followed by data extraction and quantification using intensities, and extensive data analyses. (b) Total number of proteins identified (upper panel) per individual sample and distribution of normalized protein intensities (lower panel) for each sample (n = 67), showing a highly similar protein identification number between the samples and individuals. (c) Data presence plot, showing a high data presence amongst…

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  Figure 2. Personal and inter-individual variation of the urinary EV proteome. (a) Unsupervised spearman correlation analysis of 8 individuals (67 samples) based on total proteome (1802 proteins) with the correlation coefficient values from 0 (white) to 1 (red). (b) Hierarchical clustering of the top 10% most variable urinary EV proteins (180 proteins) showing a clustering largely based on individual. (c) Number of proteins identified at all timepoints (purple), at more than 1 timepoint (blue) or at a single timepoint (gray) per donor. Percentages relative to the total proteins identified per person are annotated on the barplots. (d) Number of proteins identified in all individuals (core proteome, 516 proteins), in more than 1 individual (total 1174 proteins), and unique proteins per person (combined 113 proteins). (e) Distribution of personal CVs (intra-CVs) of each individual for the core urinary EV proteome (516 proteins) which was defined as the proteins detected at all timepoints in all 8 individuals. (f) Distribution of inter- and intra-CVs for the core urinary EV proteome (516 proteins). Inter-CV is calculated amongst 67 samples, and intra-CV is represented as the mean of the…

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  Figure 3. Enriched biological functions in the core urinary EV proteome. (a) Gene ontology (GO) term cellular component (CC) analysis of the core urinary EV proteome. Barplots show top 5 GO:CC terms associated with the core urinary EV proteome, analyzed using ClueGO28. (b) Gene ontology (GO) term biological process (BP) analysis of the core urinary EV proteome. Barplots show top 5 GO:BP terms associated with the core urinary EV proteome, analyzed using ClueGO28. (c) Schematic overview of the enriched biological functions in the core urinary EV protein network (516 proteins), analyzed by clusterONE31 and BINGO32. For the detailed network, see Supplementary Fig. 4. (d) Protein interaction network of EV markers and associated signaling and localization protein clusters, predicted by clusterONE31. (e) Protein interaction network of metabolic cluster. Enriched sub-networks predicted by clusterONE31 are indicated within the figure. PPP: Pentose-Phosphate Pathway.

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  Figure 4. Gender-based differences in the urinary EV proteome. (a) Gene set enrichment analysis (GSEA, hallmarks) of the differential analysis between female- and male-derived urinary EVs was performed by ranking proteins based on the sign of their fold change and p-value, with proteins significantly overexpressed in females at the top of the list. Gene sets enriched in females are marked pink, and gene sets enriched in males are marked blue. The size of the dot reflects the significance of the enrichment (false discovery rate). (b) Volcano plot of the urinary EV protein expression levels between female and male samples. Colored dots indicate the 172 proteins that were significantly different between female or male samples. A total of 89 proteins were upregulated in males (left side, in blue), whereas 83 proteins were overexpressed in females (right side, pink). Proteins-of-interest that are shown in Fig. 4C are annotated in the plot. (c) Expression levels of selected female- (upper 2 panels) and male-specific (lower 2 panels) urinary EV proteins, showing an enrichment of gender-specific proteins in the expected samples. (d) Protein interaction networks enriched in female (left) and male…