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Urine provides a diverse source of information related to health status and is ideal for clinical proteomics due to its ease of collection. To date, there is no standard operating procedure for reproducible and robust urine sample preparation for mass spectrometry-based clinical proteomics. To this end, a novel workflow was developed based on an on-bead protein capture, clean up, and digestion without the requirement for processing steps such as precipitation or centrifugation. The workflow was applied to an acute kidney injury (AKI) pilot study. Urine from clinical samples and a pooled sample were subjected to automated sample preparation in a KingFisher™ Flex magnetic handling station using a novel urine-HILIC (uHLC) approach based on MagReSyn® HILIC microspheres. For benchmarking, the pooled sample was also prepared using a published protocol based on an on-membrane (OM) protein capture and digestion workflow. Peptides were analysed by LCMS in data independent acquisition (DIA) mode using a Dionex Ultimate 3000 UPLC coupled to a Sciex 5600 mass spectrometer. Data was searched in Spectronaut™ 17. Both workflows showed similar peptide and protein identifications in the pooled sample. The uHLC workflow was easier to set up and complete, having less hands-on time than the OM method, with fewer manual processing steps. Lower peptide and protein CV was observed in the uHLC technical replicates. Following statistical analysis, candidate protein markers were filtered, at ≥ 2-fold change in abundance, ≥ 2 unique peptides and ≤ 1% false discovery rate, and revealed many significant, differentially abundant kidney injury-associated urinary proteins. The pilot data derived using this novel workflow provides information on the urinary proteome of patients with AKI. Further exploration in a larger cohort using this novel high-throughput method is warranted.

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