System-Wide Profiling of Protein Amino Termini from Formalin-Fixed, Paraffin-Embedded Tissue Specimens for the Identification of Novel Substrates.
Clinical tissues are used for histopathological diagnosis of many diseases including immunostaining and morphology subtyping as well as in molecular research such as for the analyses of DNA, RNA, and proteins. Formalin fixation and paraffin embedment (FFPE) of tissue specimens is routinely used for preserving clinical tissues for long-term storage, allowing histopathological diagnosis of many diseases. As such, FFPE tissues currently represent the most comprehensive collection of all clinical specimens, allowing great source of material for research opportunity, possibly due to the concern of protein integrity from antigen retrieval from fixation process. Hence, to date, very few studies have used FFPE specimens to look at the profiling of protein termini. Nevertheless in the field of protease research, the protein amino termini are particularly useful for the system-wide identification of substrates and for the characterization of protease-mediated cleavage sites . In this chapter, we outline a robust methodology for the extraction of proteins from FFPE specimens for the enrichment of protein amino termini. This approach enriches endogenous protein N-termini by removal of internal peptides using synthetic polymers of hyperbranched polyglycerol aldehyde. As a result, protein amino termini are analyzed using mass spectrometers to elucidate the biological regulation of protease-substrate interactions in healthy and diseased tissues.
Department of Genetics and Complex Diseases, Harvard T. H. Chan School of Public Health, 655 Huntington Avenue, Boston, MA, 02115, USA.|Institute of Molecular Medicine and Cell Research, University of Freiburg, Stefan-Meier-Strasse 17, 79104, Freiburg, Germany.
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Institute of Molecular Medicine and Cell Research, University of Freiburg, Stefan-Meier-Strasse 17, 79104, Freiburg, Germany. oliver.schilling@mol-med.uni-freiburg.de.|BIOSS Centre of Biological Signaling Studies, University of Freiburg, 79104, Freiburg, Germany. oliver.schilling@mol-med.uni-freiburg.de.|German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany. oliver.schilling@mol-med.uni-freiburg.de.
Oliver
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Schilling
D002853:Chromatography, Liquid / Q000379:methods; D010446:Peptide Fragments; D010447:Peptide Hydrolases / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D059748:Proteolysis; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D012984:Software; D013223:Statistics as Topic; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods; D057188:Workflow
D010446:Peptide Fragments; D011506:Proteins; D020543:Proteome; D010447:Peptide Hydrolases
Degradome; FFPE; Protease; Proteomics; Substrate