Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry.
A satisfactory protocol of protein extraction has been established based on the heat-induced antigen retrieval (AR) technique widely applied in immunohistochemistry for archival formalin-fixed, paraffin-embedded (FFPE) tissue sections. Based on AR, an initial serial experiment to identify an optimal protocol of heat-induced protein extraction was carried out using FFPE mouse tissues. The optimal protocol for extraction of proteins was then performed on an archival FFPE tissue of human renal carcinoma. FFPE sections were boiled in a retrieval solution of Tris-HCl containing 2% SDS, followed by incubation. Fresh tissue taken from the same case of renal carcinoma was processed for extraction of proteins by a conventional method using radioimmunoprecipitation assay solution, to compare the efficiency of protein extraction from FFPE tissue sections with extraction from fresh tissue. As a control, further sections of the same FFPE sample were processed by the same procedure without heating treatment. Evaluation of the quality of protein extracted from FFPE tissue was done using gel electrophoresis and mass spectrometry, showing most identified proteins extracted from FFPE tissue sections were overlapped with those extracted from fresh tissue.
Shi
Shan-Rong
SR
Department of Pathology, University of Southern California Keck School of Medicine HMR 204, 2011 Zonal Avenue, Los Angeles, CA 90033, USA. taylor@pathfinder.hsc.usc.edu
Liu
Cheng
C
Balgley
Brian M
BM
Lee
Cheng
C
Taylor
Clive R
CR
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005404:Fixatives; D005557:Formaldehyde; D006361:Heating; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007680:Kidney Neoplasms / Q000737:chemistry / Q000648:ultrastructure; D008099:Liver / Q000737:chemistry; D042361:Mammary Glands, Human / Q000737:chemistry; D013058:Mass Spectrometry; D051379:Mice; D016612:Paraffin Embedding; D011506:Proteins / Q000302:isolation & purification*; D011786:Quality Control
D005404:Fixatives; D011506:Proteins; D005557:Formaldehyde