title: Formalin-Fixed, Paraffin-Embedded Tissues (FFPE) as a Robust Source for the Profiling of Native and Protease-Generated Protein Amino Termini.
abstract: Dysregulated proteolysis represents a hallmark of numerous diseases. In recent years, increasing number of studies has begun looking at the protein termini in hope to unveil the physiological and pathological functions of proteases in clinical research. However, the availability of cryopreserved tissue specimens is often limited. Alternatively, formalin-fixed, paraffin-embedded (FFPE) tissues offer an invaluable resource for clinical research. Pathologically relevant tissues are often stored as FFPE, which represent the most abundant resource of archived human specimens. In this study, we established a robust workflow to investigate native and protease-generated protein N termini from FFPE specimens. We demonstrate comparable N-terminomes of cryopreserved and formalin-fixed tissue, thereby showing that formalin fixation/paraffin embedment does not proteolytically damage proteins. Accordingly, FFPE specimens are fully amenable to N-terminal analysis. Moreover, we demonstrate feasibility of FFPE-degradomics in a quantitative N-terminomic study of FFPE liver specimens from cathepsin L deficient or wild-type mice. Using a machine learning approach in combination with the previously determined cathepsin L specificity, we successfully identify a number of potential cathepsin L cleavage sites. Our study establishes FFPE specimens as a valuable alternative to cryopreserved tissues for degradomic studies.
authors.affiliation: From the ‡Institute of Molecular Medicine and Cell Research.
authors.forename: Zon Weng
authors.initials: ZW
authors.lastname: Lai
authors.affiliation: From the ‡Institute of Molecular Medicine and Cell Research.
authors.forename: Juliane
authors.initials: J
authors.lastname: Weisser
authors.affiliation: From the ‡Institute of Molecular Medicine and Cell Research.
authors.forename: Lars
authors.initials: L
authors.lastname: Nilse
authors.affiliation: §Department of Computer Science.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Costa
authors.affiliation: From the ‡Institute of Molecular Medicine and Cell Research.
authors.forename: Eva
authors.initials: E
authors.lastname: Keller
authors.affiliation: From the ‡Institute of Molecular Medicine and Cell Research.
authors.forename: Martina
authors.initials: M
authors.lastname: Tholen
authors.affiliation: ¶Department of Pathology and Laboratory Medicine and Department of Chemistry, Centre of Chemistry, University of British Columbia, Vancouver, Canada;
authors.forename: Jayachandran N
authors.initials: JN
authors.lastname: Kizhakkedathu
authors.affiliation: From the ‡Institute of Molecular Medicine and Cell Research.
authors.forename: Martin
authors.initials: M
authors.lastname: Biniossek
authors.affiliation: ‖Department of Pathology, **German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Bronsert
authors.affiliation: From the ‡Institute of Molecular Medicine and Cell Research, **German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany ‡‡BIOSS Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany; oliver.schilling@mol-med.uni-freiburg.de.
authors.forename: Oliver
authors.initials: O
authors.lastname: Schilling
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid; D015925:Cryopreservation; D008099:Liver / Q000378:metabolism*; D000069550:Machine Learning; D051379:Mice; D016612:Paraffin Embedding; D010447:Peptide Hydrolases / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry*; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D016707:Tissue Fixation
chemical_list: D011506:Proteins; D010447:Peptide Hydrolases