Urine sample preparation in 96-well filter plates for quantitative clinical proteomics

Area of Science:

• Proteomics
• Biomarker Discovery
• Analytical Chemistry

Background:

• Urine is a valuable, non-invasive source for disease diagnosis and prognosis.
• Liquid chromatography-mass spectrometry (LC-MS) proteomics is a sensitive method for identifying urinary disease biomarkers.
• Current methods require optimization for high-throughput clinical applications.

Purpose of the Study:

• To develop and validate a robust, high-throughput filter-aided sample preparation (FASP) method for urine proteomics.
• To adapt FASP to a 96-well plate format for parallel processing.
• To assess the method's performance in terms of protein identification, repeatability, and applicability to different urine sample types.

Main Methods:

• Development of a 96-well plate-based FASP protocol (96FASP).
• Processing of soluble urine concentrates (~10 μg total protein) and urinary pellet lysates using 96FASP.
• Analysis by LC-MS and label-free quantification.
• Assessment of experimental repeatability using Pearson correlation analysis.

Main Results:

• 96FASP identified 700-900 proteins from soluble urine concentrates with high repeatability (R ≥ 0.97).
• Analysis of urinary pellet lysates yielded an average of 1700 proteins per experiment.
• Pilot study demonstrated distinct protein profiles between different urine donors and clustering of technical replicates.

Conclusions:

• The 96FASP method offers a robust and cost-effective approach for high-throughput urine proteomics.
• This method facilitates efficient peptide generation from urine for biomarker discovery.
• 96FASP has the potential to become a standard for large-scale clinical proteomics projects.