Mass spectrometry-based urinary proteomics is one of the most attractive strategies to discover
proteins for diagnosis, prognosis, monitoring, or prediction of therapeutic responses of
urological diseases involving the kidney, prostate, and bladder; however, interfering compounds found in urine necessitate sample preparation strategies that are currently not suitable for urinary proteomics in the clinical setting. Herein, we describe the C4-tip method, comprising a simple, automated strategy utilizing a reverse-phase resin tip-based format and "on-tip" digestion to examine the urine
proteome. We first determined the optimal conditions for
protein isolation and
protease digestion on the C4-tip using the standard
protein bovine
fetuin. Next, we applied the C4-tip method to urinary proteomics, identifying a total of 813
protein groups using LC-MS/MS, with identified
proteins from the C4-tip method displaying a similar distribution of gene ontology (GO) cellular component assignments compared to identified
proteins from an ultrafiltration preparation method. Finally, we assessed the reproducibility of the C4-tip method, revealing a high Spearman correlation R-value for shared
proteins identified across all
tips. Together, we have shown the C4-tip method to be a simple, robust method for high-throughput analysis of the urinary
proteome by mass spectrometry in the clinical setting.