title: Proteomic sample preparation from formalin fixed and paraffin embedded tissue.
abstract: Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.
authors.affiliation: Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry.
authors.forename: Jacek R
authors.initials: JR
authors.lastname: Wiśniewski
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D005557:Formaldehyde / Q000737:chemistry*; D006801:Humans; D042282:Microdissection / Q000379:methods; D008867:Microtomy / Q000379:methods; D016612:Paraffin Embedding / Q000379:methods*; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D010455:Peptides; D005557:Formaldehyde
references.citation: Mol Syst Biol. 2011 Nov 08;7:548
references.pmid: 22068331
references.citation: J Proteome Res. 2009 Dec;8(12):5674-8
references.pmid: 19848406
references.citation: Mol Syst Biol. 2012;8:611
references.pmid: 22968445
references.citation: Anal Chem. 2012 Mar 20;84(6):2631-7
references.pmid: 22324799
references.citation: Proteomics Clin Appl. 2013 Apr;7(3-4):225-33
references.pmid: 23090905
references.citation: J Proteome Res. 2011 Jul 1;10(7):3040-9
references.pmid: 21526778
references.citation: Mol Syst Biol. 2011 Nov 08;7:549
references.pmid: 22068332
references.citation: Proteomics. 2012 Apr;12(7):1045-58
references.pmid: 22318899
references.citation: Proteomics Clin Appl. 2013 Apr;7(3-4):241-51
references.pmid: 23027712
references.citation: Nat Methods. 2009 May;6(5):359-62
references.pmid: 19377485
references.citation: Anal Chem. 2003 Feb 1;75(3):663-70
references.pmid: 12585499
references.citation: J Proteome Res. 2010 Jul 2;9(7):3688-700
references.pmid: 20469934