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.

Hope-Bal

“…Furthermore, a variety of commonly used ICC markers can be applied for diagnostic analysis. It was previously shown (Kähler et al 2010) that the HOPE technique allows two-dimensional separation of proteins in paraffin-embedded tissue samples, with further validation by mass spectrometry and protein Asthma 5645 643 21 6309 3989 5051 969 10,009 COPD 5895 945 10 6850 3629 4726 1800 10,155 Emphysema 3899 1158 40 5097 4418 1416 38 5872 Fibrosis 2833 687 39 3559 2644 571 56 3271 Sarcoidosis 2588 535 31 3154 2009 594 46 2649 Tuberculosis 3038 842 29 3909 2227 488 57 2772 COPD, chronic obstructive pulmonary disease.…”

KINFix – A formalin-free non-commercial fixative optimized for histological, immunohistochemical and molecular analyses of neurosurgical tissue specimens

“…Fresh frozen tissue is the gold standard for such purposes, however, the preparation and storage of samples at –70 °C is elaborate, expensive and not always possible (e.g., small biopsy specimens cannot be split into two parts, and professional laboratory staff is usually not available in the evening and on weekends). Furthermore, the destruction of fine tissue morphology significantly decreases the spatial resolution, while the degradation of nucleic acids cannot be entirely avoided at long-term storage [ 23 ].…”

The tissue is the issue: improved methylome analysis from paraffin-embedded tissues by application of the HOPE technique

“…18,26 The HOPE technique 10 is a preservation technique that leads to paraffin-embedded tissues without cross-linking of proteins 13 and only little degradation of nucleic acids. 11 Recently it has been shown that HOPE-fixed biomaterial allows a multitude of different molecular approaches such as 2D electrophoresis 7 or transcriptome analysis 29 out of the very same paraffin block. As the tissue processing takes 1 day longer than classical FFPE material, this might raise objections for application in routine diagnostics work-flows and therefore favors its use for biobanking or researchorientated approaches.…”

“…Therefore, investigators have to face a trade-off between the applicability of different techniques with respect to both, the available amount of material and the way of tissue preservation. Recent developments have successfully demonstrated the feasibility of applying diverse techniques such as 2D proteome analysis 7 or qPCR and transcriptome analysis 8,9 from the same paraffin-embedded specimen block by application of the HOPE fixation. 10 Using HOPE for biomaterial preservation offers a multitude of read-out techniques with little nucleic acid degradation 11,12 and the possibility of immunophenotyping the sample without epitope-retrieval.…”