Simple, Fast, and Highly Efficient One- or Two-Step Proteomic Preparation Enables Deep Profiling of Microgram-Level...

Area of Science:

• Biochemistry
• Proteomics
• Analytical Chemistry

Background:

• Large-scale tissue proteomics demands efficient, rapid, and repeatable workflows.
• Existing methods often face challenges with diverse sample types like fresh-frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) tissues.
• Deep proteome coverage and high repeatability are crucial for translational research.

Purpose of the Study:

• To develop and present a Simple Workflow for Integrated and Fast Tissue-preparation (SWIFT).
• To enable efficient and rapid processing of both FF and FFPE tissues for deep proteomics.
• To demonstrate high repeatability and broad applicability across various tissue types and sample amounts.

Main Methods:

• SWIFT integrates lysis, reduction, alkylation, and digestion for FF tissues in ≤1.5 hours.
• FFPE tissues undergo concurrent deparaffinization, rehydration, and de-cross-linking in 0.5 hours, followed by one-step peptide preparation.
• The workflow eliminates desalting and offline cleanup steps to reduce variability and processing time.

Main Results:

• Identified up to ~10,000 protein groups and ~150,000 peptides across mouse organs using timsTOF Pro.
• Achieved high repeatability (pairwise Pearson's r > 0.96) across six replicates with 6-7 orders of magnitude dynamic range.
• Revealed preservation-induced proteome shifts, with FFPE tissues showing reduced detection of membrane and respiratory proteins.

Conclusions:

• SWIFT provides a fast, simplified, and repeatable approach for deep tissue proteomics.
• The workflow is cost-effective and accessible for large-scale clinical and translational studies.
• Enables comprehensive proteome analysis from low- to microgram-level tissue samples.