Deep proteome mining of FFPE tissue with PASEF technology
Formaldehyde fixation and paraffin embedded (FFPE) tissues are ubiquitous inFormaldehyde fixation and paraffin embedded (FFPE) tissues are ubiquitous inhistological archives and invaluable clinical resources for diagnosis, treatment, andcharacterization of disease. However, FFPE tissue is a persistent proteomic challenge.Here, we define a robust, reproducible method by exploring orthogonal parametersefficiently. Of note, we tested hydrophilic interaction chromatography (HILIC) toenrich peptides from FFPE tissue following proteolytic digest in the presence ofdetergents and contaminants against more traditional C18-based enrichment.Further, we investigated use of the Barocycler from Pressure Biosciences Inc. (PBI) forhigh efficiency protein extraction and digestion. We also compare two different MStechnologies, timsTOF with parallel accumulation serial fragmentation (PASEF)and Orbitrap (Fusion) to determine the advantages of orthogonal gas-phaseseparations. We observe increased peptide and protein depth by timsTOF with PASEF,at least in part due to the sensitivity and analysis speed of the instrument. Overall,we present our recommendations for robust analysis of tissues to open doors toinformation previously hidden in the large tranche of FFPE archives.