Simple Tip-Based Sample Processing Method for Urinary Proteomic Analysis

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.