Generation of high-quality protein extracts from formalin-fixed, paraffin-embedded tissues.
A wealth of information on proteins involved in many aspects of disease is encased within formalin-fixed paraffin-embedded (FFPE) tissue repositories stored in hospitals worldwide. Recently, access to this "hidden treasure" is being actively pursued by the application of two main extraction strategies: digestion of the entangled protein matrix with generation of tryptic peptides, or decrosslinking and extraction of full-length proteins. Here, we describe an optimised method for extraction of full-length proteins from FFPE tissues. This method builds on the classical "antigen retrieval" technique used for immunohistochemistry, and allows generation of protein extracts with elevated and reproducible yields. In model animal tissues, average yields of 16.3 microg and 86.8 microg of proteins were obtained per 80 mm(2) tissue slice of formalin-fixed paraffin-embedded skeletal muscle and liver, respectively. Protein extracts generated with this method can be used for the reproducible investigation of the proteome with a wide array of techniques. The results obtained by SDS-PAGE, western immunoblotting, protein arrays, ELISA, and, most importantly, nanoHPLC-nanoESI-Q-TOF MS of FFPE proteins resolved by SDS-PAGE, are presented and discussed. An evaluation of the extent of modifications introduced on proteins by formalin fixation and crosslink reversal, and their impact on quality of MS results, is also reported.
Addis
Maria Filippa
MF
Porto Conte Ricerche Srl, Tramariglio, Alghero, Sassari, Italy. addis@portocontericerche.it
Tanca
Alessandro
A
Pagnozzi
Daniela
D
Crobu
Salvatore
S
Fanciulli
Giuseppe
G
Cossu-Rocca
Paolo
P
Uzzau
Sergio
S
D000818:Animals; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005404:Fixatives; D005557:Formaldehyde; D016612:Paraffin Embedding / Q000379:methods*; D040081:Protein Array Analysis; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification; D015203:Reproducibility of Results; D012756:Sheep; D053719:Tandem Mass Spectrometry; D016707:Tissue Fixation / Q000379:methods*
D005404:Fixatives; D011506:Proteins; D005557:Formaldehyde