title: Peptide extraction from formalin-fixed paraffin-embedded tissue.
abstract: This unit describes how to extract tryptic peptides from formalin-fixed, paraffin-embedded (FFPE) tissues for analysis using nano-reverse-phase liquid chromatography/tandem mass spectrometry (nRPLC-MS/MS). The tissues are deparaffinized in the first protocol. Following deparaffinization, the cells are harvested via one of two methods: needle dissection or laser capture microdissection (LCM). Needle dissection is performed using hydrated, unstained tissue, whereas LCM is performed with dehydrated, hematoxylin-stained tissue. Heat is applied to the collected cells to reverse the cross-links that have formed during the formalin fixation process. Finally, the cells are digested using filter-aided sample preparation, in which buffers are exchanged throughout the process. An alternate protocol using commercially available Liquid Tissue is also described. These samples are then ready for mass spectrometric analysis.
authors.affiliation: Phoenix S&T, Chester, Pennsylvania, USA.
authors.forename: Katherine J
authors.initials: KJ
authors.lastname: Heaton
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Master
mesh_terms: D002469:Cell Separation; D056148:Chromatography, Reverse-Phase; D005557:Formaldehyde; D006651:Histocytochemistry; D006358:Hot Temperature; D042282:Microdissection / Q000379:methods*; D010232:Paraffin / Q000302:isolation & purification; D016612:Paraffin Embedding; D010455:Peptides / Q000032:analysis / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D010455:Peptides; D005557:Formaldehyde; D010232:Paraffin