Issue 13, 2024

An effective urobilin clearance strategy based on paramagnetic beads facilitates microscale proteomic analysis of urine

Abstract

Urine provides an ideal source for disease biomarker discovery. High-adhesion contaminants such as urobilin, which are difficult to remove from urine, can severely interfere with urinary proteomic analysis. Here, we aimed to establish a strategy based on single-pot, solid-phase-enhanced sample preparation (SP3) technology to prepare samples for urinary proteomics analysis that almost completely eliminates the impact of urobilin. A systematic evaluation of the effects of two urinary protein precipitation methods, two types of protein lysis buffers, and different ratios of magnetic digestion beads on the identification and quantification of the microscale urinary proteome was conducted. Our results indicate that methanol–chloroform precipitation, coupled with efficient lysis facilitated by urea, and subsequent enzymatic digestion using a mix of hydrophilic and hydrophobic magnetic beads offers the best performance. Further applying this strategy to the urine of patients with benign prostatic hyperplasia, prostate cancer and healthy individuals, combined with a narrow window of data-independent acquisition, FGFR4, MYLK, ORM2, GOLM1, SPP1, CD55, CSF1, DLD and TIMP3 were identified as potential biomarkers to discriminate benign prostatic hyperplasia and prostate cancer patients.

Graphical abstract: An effective urobilin clearance strategy based on paramagnetic beads facilitates microscale proteomic analysis of urine

Supplementary files

Article information

Article type
Paper
Submitted
27 Feb 2024
Accepted
16 May 2024
First published
20 May 2024

Analyst, 2024,149, 3625-3635

An effective urobilin clearance strategy based on paramagnetic beads facilitates microscale proteomic analysis of urine

K. Zhen, W. Hou, L. Bai, M. Wang, Z. Yue, Z. Xu, D. Xiong, L. Gao and W. Ying, Analyst, 2024, 149, 3625 DOI: 10.1039/D4AN00312H

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