Heat 'n Beat: A Universal High-Throughput End-to-End Proteomics Sample Processing Platform in under an Hour.

Area of Science:

• Proteomics
• Mass Spectrometry
• Biochemistry

Background:

• High-throughput proteomic analysis of small solid tissue samples (≤2 mg) demands comprehensive coverage.
• Existing methods face challenges with diverse sample formats and require extensive preparation.
• Need for a rapid, robust, and universal sample preparation protocol for mass spectrometry.

Purpose of the Study:

• To develop a universal, rapid, and robust sample preparation protocol for high-throughput proteomic analysis.
• To optimize a one-tube homogenization and digestion method for diverse tissue types and fixation methods.
• To demonstrate the protocol's efficiency, yield, and consistency across various biological samples.

Main Methods:

• Developed the Heat 'n Beat (HnB) one-tube homogenization and digestion method.
• Adapted sample washing and peptide cleanup for fresh frozen (FF), FF-OCT, and FFPE tissues.
• Optimized tissue disruption, lysis, reduction, alkylation, and proteolysis into a single, rapid step.

Main Results:

• HnB protocol significantly increased peptide yield compared to previous methods.
• Achieved high digestion efficiency (85-90%) with core processing in 38 minutes (total 53-63 minutes).
• Demonstrated robustness on six organ types, a cell line, and a cancer biopsy.
• Successfully processed 1171 FF-OCT human cancer biopsies in 92 hours, yielding consistent results for 3513 MS runs.

Conclusions:

• The Heat 'n Beat (HnB) protocol provides a universal, rapid, and robust solution for high-throughput tissue proteomic analysis.
• The method is applicable to various tissue types and fixation methods, enhancing proteome coverage and efficiency.
• HnB significantly streamlines sample preparation, enabling large-scale proteomic studies with consistent, high-quality data.