Sample Preparation for High-Throughput Urine Proteomics Using 96-Well Polyvinylidene Fluoride (PVDF) Membranes

Area of Science:

• Clinical Proteomics
• Biomarker Discovery
• Mass Spectrometry

Background:

• Proteomics analysis of urine is crucial for studying systemic perturbations.
• Biomarker discovery requires analyzing large patient cohorts to account for biological variability.
• High-throughput sample processing is essential for clinical proteomics to minimize bias.

Purpose of the Study:

• To describe a high-throughput method for parallel 96-well plate-based processing of urine samples.
• To provide a detailed protocol for 'MStern blotting' sample processing for mass spectrometry-based proteomics.
• To demonstrate the utility of urine proteomics in identifying protein catabolism sites and informing therapeutic strategies.

Main Methods:

• Developed a 96-well plate-based processing strategy using polyvinylidene fluoride (PVDF) membrane plates.
• Utilized vacuum manifolds and multichannel pipettes/liquid handling robots for efficient sample processing.
• Applied the 'MStern blotting' protocol to patient urine samples for LC/MS-based proteomics.

Main Results:

• Successfully implemented a high-throughput method for urine sample processing.
• Demonstrated the identification of protein catabolism sites using minimal urine volumes (150 μL).
• Showcased urine as a valuable biofluid for reflecting systemic states.

Conclusions:

• The described 'MStern blotting' method enables efficient, high-throughput urine sample processing for clinical proteomics.
• This approach facilitates biomarker discovery and the study of protein catabolism in critical illness.
• Urine proteomics offers a non-invasive and effective means to assess systemic conditions.