An analysis of the impact of pre‐analytical factors on the urine proteome: Sample processing time, temperature, and proteolysis

PURPOSE: We have examined the impact of sample processing time delay, temperature, and the addition of protease inhibitors (PIs) on the urinary proteome and peptidome, an important aspect of biomarker studies. EXPERIMENTAL DESIGN: Ten urine samples from patients with varying pathologies were each divided and PIs added to one‐half, with aliquots of each then processed and frozen immediately, or after a delay of 6 h at 4°C or room temperature (20–22°C), effectively yielding 60 samples in total. Samples were then analyzed by 2D‐PAGE, SELDI‐TOF‐MS, and immunoassay. RESULTS: Interindividual variability in profiles was the dominant feature in all analyses. Minimal changes were observed by 2D‐PAGE as a result of delay in processing, temperature, or PIs and no changes were seen in IgG, albumin, β(2)‐microglobulin, or α(1)‐microglobulin measured by immunoassay. Analysis of peptides showed clustering of some samples by presence/absence of PIs but the extent was very patient‐dependent with most samples showing minimal effects. CONCLUSIONS AND CLINICAL RELEVANCE: The extent of processing‐induced changes and the benefit of PI addition are patient‐ and sample‐dependent. A consistent processing methodology is essential within a study to avoid any confounding of the results.