Massive formalin-fixed, paraffin-embedded (FFPE) tissue archives exist worldwide, representing an invaluable resource for clinical proteomics research. However, current protocols for FFPE proteomics lack standardization, efficiency, reproducibility, and scalability. Here we present high-yield protein extraction and recovery by direct solubilization (HYPERsol), an optimized workflow using ultrasonication and S-Trap sample processing that enables proteome coverage and quantification from FFPE samples comparable to that achieved from flash-frozen tissue (average R = 0.936). When applied to archival samples, HYPERsol resulted in high-quality data from FFPE specimens in storage for up to 17 years, and may enable the discovery of new immunohistochemical markers.

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Massive formalin-fixed, paraffin-embedded (FFPE) tissue archives exist worldwide, representing a potential gold mine for clinical proteomics research. However, current protocols for FFPE proteomics lack standardization, efficiency, reproducibility, and scalability. Here we present High-Yield Protein Extraction and Recovery by direct SOLubilization (HYPER-sol), an optimized workflow using adaptive-focused acoustics (AFA) ultrasonication and S-Trap sample processing that enables proteome coverage and quantification from FFPE samples comparable to that achieved from flash-frozen tissue (average R = 0.936). BodyFormalin fixation and paraffin embedding (FFPE) is a tissue preparation method common in experimental research and medicine. It is standard in all pathology departments where pathological diagnosis is based on tissue section staining and immunohistochemistry on FFPE slides. The method is over one hundred years old and yields biologically inactive samples that are stable at room temperature for decades and longer 1-3 . The ubiquity of this practice in pathology combined with the unique stability of FFPE samples has resulted in massive numbers of specimens housed in countless historical tissue archives around the world. These collections represent an invaluable treasure-trove for retrospective research and translational studies, especially when specimens are paired with medical records describing the diagnosis and course of disease. However, despite this huge potential, proteomic analysis of FFPE samples has yet to become mainstream 4 . Multiple disparate protocols for proteomic analysis of FFPE material exist and generally entail a laborious deparaffinization process requiring multiple changes of toxic solvents. More significantly, despite its essentiality to proteomics analysis, no consensus has been reached on an optimal method for protein extraction 5 .Here we present High-Yield Protein Extraction and Recovery by direct SOLubilization (HYPER-sol), a standardized workflow for FFPE proteomics which combines AFA TM ultrasonication and S-Trap TM 6 sample processing to permit highly similar peptide and protein identifications to those achieved from paired flash-frozen tissue ("F"). We developed HYPER-sol by optimizing the techniques of deparaffinization, protein solubilization, and sample preparation to peptides for proteomics analysis. We compared the standard xylene-ethanol deparaffinization procedure ("X") to a new procedure in which FFPE cores were directly solubilized in buffer containing 5% SDS ("D"). Protein extraction was performed with either probe sonication ("P") or Covaris AFA ultrasonication ("A"). Protein was recovered and processed for liquid chromatography-mass spectrometry with either Wessel-Flügge methanol-chloroform precipitation 7 and in-solution digestion ("M") or with S-Traps ("S") [ Fig 1a].We first examined the extent of solubilization of FFPE liver samples achieved by each method. Compared to the standard workflow employing xylene-ethanol deparaffinization and probe sonication ("XP") 8 , ...