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

ABSTRACT Large-scale tissue proteomics requires workflows that are efficient, rapid, and repeatable across diverse samples. Herein, we present Simple Workflow for Integrated and Fast Tissue-preparation (SWIFT), which enables complete processing of fresh- frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) tissues in either one- or two-step formats, while maintaining deep proteome coverage with high repeatability from low to microgram-level tissues. For FF tissues, an incubation process integrating lysis, reduction, alkylation, and digestion generates peptide samples directly from tissues in ≤ 1.5 h. For FFPE tissues, concurrent deparaffinization, rehydration, and de- crosslinking is achieved within 0.5 h, followed by one-step peptide preparation. Furthermore, our workflows eliminate desalting and offline cleanup steps, thereby reducing variability and total processing time. Using our methods, we identified up to ∼10,000 protein groups and ∼150,000 peptides across multiple mouse organs on timsTOF Pro. Repeatability was high (pairwise Pearson’s r > 0.96 across six experimental replicates), with dynamic ranges spanning 6–7 orders of magnitude. Organ-enriched protein analysis identified functionally distinct proteins unique to each tissue Paired FF and FFPE analyses revealed preservation-induced shifts, with FFPE tissues showing reduced detection of membrane-associated and respiratory proteins, including mitochondrial Complex I. Together, our fast and simplified workflows enable deep tissue proteomics for large-scale clinical and translational studies in a cost- effective and widely-accessible manner.