Characterization of the Urine Proteome Using the Olink Explore 3072 Platform

Abstract Urinary proteins are valuable biomarkers for physiological processes and early detection and monitoring of systemic and kidney diseases. However, comprehensive quantification and characterization of the urine proteome using high-throughput platforms remains limited. Using an affinity-based assay for large-scale proteomics, the Olink Explore 3072 platform, we quantified 2,868 proteins in urine samples from 1,268 participants of the German Chronic Kidney Disease (GCKD) study. Protein detectability in urine, defined as the percentage of samples with measurements above the limit of detection, showed a bimodal distribution: 659 (23%) proteins had high (>80%) and 1,336 proteins (47%) had low (<20%) detectability. Over 95% of highly detectable proteins were confirmed as present in urine by mass spectrometry. They were enriched for extracellular, exosomal, and cell-surface localization, and showed tissue-specific expression in hepatocytes, immune cells, and proximal tubule cells. Significant associations of protein levels with sex (921 proteins), BMI (367 proteins) and age (157 proteins) showed plausible relationships such as prostate-specific antigen and male sex. Unsupervised correlation analysis of protein levels detected multiple biologically meaningful protein clusters, reflecting shared cellular compartments (e.g., lysosomal origin), tissue of origin (e.g., liver), and molecular interactions (e.g., receptor-ligand or chaperone-transporter complexes). In conclusion, the Olink Explore 3072 platform is well-suited for large-scale urine proteomics and identifies biologically plausible representations of ongoing processes in health and disease. Our comprehensive characterization provides a valuable resource for biomarker discovery and future targeted studies of urine proteins. Translational Statement Urinary proteins reflect kidney-specific and systemic processes, with the potential to serve as disease biomarkers. We demonstrate that the Olink Proximity Extension Assay enables high-throughput urine proteome screening with sufficient specificity to identify biologically plausible protein signatures and demographic associations. By providing comprehensive data on protein detectability, variability, and clinical associations, our study serves as a reference resource for designing targeted proteomic investigations, facilitating the development of urine-based biomarker panels for different purposes, potentially including early detection of impaired kidney function, monitoring kidney disease progression, and predicting treatment response.