Proteomics out of the archive: Two-dimensional electrophoresis and mass spectrometry using HOPE-fixed, paraffin-embedded tissues

Abstract

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.

Figure 1

Hematoxylin/eosin -stained sections from all investigated tissue samples. (A, C–E) Adenocarcinomas; (B,F) squamous cell carcinomas; (G) tumor-free lung. H, HOPE-fixed tissue; FR, frozen; FF, formalin-fixed; ET, ethanol-fixed. Bar = 100 μm.

Figure 2

Comparison of 2DE results from adenocarcinoma (A) and squamous cell carcinoma (B) subjected to aqueous protein extraction. H, HOPE-fixed; FR, frozen; FF, formalin-fixed.

Figure 3

Comparison of 2DE results from three adenocarcinomas (C–E) subjected to complex protein extraction. H, HOPE-fixed; FR, frozen material; FF, formalin-fixed.

Figure 4

Comparison of 2DE results from one adenocarcinoma (F) with those from tumor-free lung tissue (G). H, HOPE-fixed; FF, formalin-fixed; ET, ethanol-fixed.

Figure 5

Squamous cell carcinoma subjected to Western immunoblot targeting pan-keratin and the corresponding twin gel which was used for the tryptic digests. Enlarged section shows the exact positions of the chosen spots; the results of the mass spectrometric protein identification are shown in the lower right.

Figure 6

Mascot search results with probability-based MOWSE scores of four protein spots excised from HOPE-derived 2DE. (A) Keratin 10; (B) human serum albumin; (C) tropomyosin; (D) calreticulin.