Proteomics out of the archive: Two-dimensional electrophoresis and mass spectrometry using HOPE-fixed, paraffin-embedded tissues.
Proteome analyses provide diagnostic information which can be essential for therapeutic predictions. The application of such techniques for analyzing paraffin-embedded tissue samples is widely hampered by the use of formalin fixation requiring antigen retrieval procedures in molecular pathology. In prior studies, the HEPES-glutamic acid buffer-mediated organic solvent protection effect (HOPE) technique of tissue fixation has been shown to provide a broad array of biochemical investigations with excellent preservation of morphological structures, DNA, RNA, and proteins, thus supporting the multimethod analysis of archived specimens. Here we show that HOPE fixation is also useful in proteomic investigations by allowing two-dimensional electrophoresis (2DE) and mass spectrometry, using lung cancer tissues. Two-dimensional gels of two-protein extraction protocols derived from HOPE-fixed material displayed characteristic spot patterns with high reproducibility. For comparison, 2DE analysis of ethanol-fixed, formalin-fixed, and frozen samples from the same tissues was performed. Western blotting confirmed immunoreactivity of 2DE-separated proteins from HOPE-fixed tissue samples. Additionally, distinct spots were excised from HOPE-derived 2D gels and successfully subjected to peptide mass fingerprinting. In conclusion, paraffin archives containing HOPE-fixed tissues are applicable to a wide spectrum of molecular investigations including common biochemical methods for proteome analyses and therefore represent a unique source for molecular investigations in the rapidly growing field of molecular pathology. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
Kähler
Daniel
D
Research Center Borstel, Germany. dkaehler@fz-borstel.de
Alexander
Christian
C
Schultz
Holger
H
Abdullah
Mahdi
M
Branscheid
Detlev
D
Lindner
Buko
B
Zabel
Peter
P
Vollmer
Ekkehard
E
Goldmann
Torsten
T
D000230:Adenocarcinoma / Q000737:chemistry*; D014408:Biomarkers, Tumor / Q000032:analysis*; D015153:Blotting, Western; D002289:Carcinoma, Non-Small-Cell Lung / Q000737:chemistry*; D002294:Carcinoma, Squamous Cell / Q000737:chemistry*; D015925:Cryopreservation; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008175:Lung Neoplasms / Q000737:chemistry*; D016612:Paraffin Embedding; D020543:Proteome / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D016707:Tissue Fixation / Q000379:methods
D014408:Biomarkers, Tumor; D005404:Fixatives; D020543:Proteome; D005557:Formaldehyde; D000431:Ethanol