Abstract
Urine is a highly accessible biofluid for clinical proteomics, but pre-analytical variability during collection, storage, and processing can compromise reproducibility. Mass spectrometry–based proteomics enables broad measurement of urinary proteins, making it suitable for evaluating how storage conditions affect proteome composition. 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 relevant for inflammatory urine samples, where immune-cell-derived and complement-associated proteins may continue to change ex vivo during delayed processing. Here, we systematically evaluated the stability of the soluble urinary proteome under clinically relevant storage conditions. Urine samples from patients with pyelonephritis were used as a clinically relevant inflammatory urine model and processed under six pre-analytical storage conditions, including immediate freezing at −80 °C, repeated freeze–thaw cycles, and incubation for 30 min or 3 h at either 4 °C or room temperature prior to freezing. High-resolution proteomics using data-independent acquisition coupled with parallel accumulation–serial fragmentation (DIA-PASEF) identified more than 4,300 protein groups across four pyelonephritis patients. 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, particularly avoidance of prolonged room-temperature storage, to reduce storage-induced changes in immune-associated urinary proteins.