Systematic assessment of soluble urine proteome stability under various storage conditions using pyelonephritis as a model

Urine is a highly accessible, non-invasive biofluid with strong potential as a clinical liquid biopsy for biomarker discovery in urinary tract diseases. Mass spectrometry–based proteomics enables the simultaneous quantification of thousands of proteins and captures disease-relevant, pharmacologically addressable phenotypes. However, clinical translation of urinary proteomics is limited by insufficient standardization of pre-analytical workflows and limited knowledge of proteome stability during storage, compromising reproducibility. This limitation is particularly critical in urinary tract infections such as pyelonephritis, where proteomic changes induced by immune and stromal cells may be further altered ex vivo as a result of sample storage conditions. Here, we systematically evaluated the stability of the soluble urinary proteome under clinically relevant storage conditions. Urine samples from pyelonephritis patients were processed under six conditions, including immediate freezing at − 80°C, repeated freeze–thaw cycles, and incubation for 30 minutes or 3 hours at either 4°C or room temperature prior to freezing. High-resolution DIA-PASEF proteomics identified more than 4,300 protein groups across four pyelonephritis patients. Comparison with published urinary proteome datasets from healthy donors and other kidney diseases revealed 1,134 proteins uniquely detected in pyelonephritis urine. Global proteome profiles were highly reproducible across all conditions (Pearson r > 0.9). In contrast, specific immune and metabolic proteins exhibited storage-dependent abundance changes, particularly after prolonged room temperature exposure, consistent with ex vivo immune activation. These results support standardized urine handling to ensure reproducible and clinically meaningful urinary proteomics.