Automated sample preparation with SP3 for low-input clinical proteomics

Area of Science:

• Clinical Proteomics
• Biomarker Discovery
• Mass Spectrometry

Background:

• High-throughput and standardized workflows are critical for clinical proteomics.
• Processing diverse sample types (e.g., FFPE tissue, blood) requires efficient methods.
• Existing methods can be time-consuming and introduce variability.

Purpose of the Study:

• To implement and validate an automated sample preparation workflow for clinical proteomics.
• To reduce hands-on time, variability, and improve reproducibility in protein quantification.
• To demonstrate the scalability and cost-effectiveness of the automated workflow.

Main Methods:

• Single-pot solid-phase-enhanced sample preparation (SP3) adapted for a liquid handling robot (autoSP3).
• Processing of tissue lysates in a 96-well format for unbiased protein purification and digestion.
• Integration with AFA (Adaptive Focused Acoustics) ultrasonication for end-to-end automated sample preparation and LC-MS analysis.

Main Results:

• AutoSP3 significantly reduces hands-on time and variability in protein quantification.
• Reproducible quantification of 500-1,000 proteins from low-input samples (100-1,000 cells).
• Successful application to FFPE pulmonary adenocarcinoma samples, recapitulating known histological growth patterns.
• Automated end-to-end workflow for 96 intact tissue samples.

Conclusions:

• AutoSP3 provides a generic, scalable, and cost-effective solution for reproducible tissue proteomics.
• The workflow minimizes manual intervention, enhancing reliability in clinical and non-clinical settings.
• This automated approach facilitates high-throughput analysis of clinical samples for biomarker discovery.