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Fowler C.B.,Washington Veterans Affairs Medical Center | O'Leary T.J.,Biomedical Laboratory Research and Development Service | Mason J.T.,Washington Veterans Affairs Medical Center
Expert Review of Proteomics | Year: 2013

Archival formalin-fixed, paraffin-embedded (FFPE) tissue and their associated diagnostic records represent an invaluable source of retrospective proteomic information on diseases for which the clinical outcome and response to treatment are known. However, analysis of archival FFPE tissues by high-throughput proteomic methods has been hindered by the adverse effects of formaldehyde fixation and subsequent tissue histology. This review examines recent methodological advances for extracting proteins from FFPE tissue suitable for proteomic analysis. These methods, based largely upon heat-induced antigen retrieval techniques borrowed from immunohistochemistry, allow at least a qualitative analysis of the proteome of FFPE archival tissues. The authors also discuss recent advances in the proteomic analysis of FFPE tissue; including liquid-chromatography tandem mass spectrometry, reverse phase protein microarrays and imaging mass spectrometry. © 2013 Informa UK Ltd.

Hudson M.B.,Emory University | Rahnert J.A.,Emory University | Zheng B.,Emory University | Woodworth-Hobbs M.E.,Emory University | And 3 more authors.
American Journal of Physiology - Cell Physiology | Year: 2014

Skeletal muscle atrophy occurs in response to a variety of conditions including chronic kidney disease, diabetes, cancer, and elevated glucocorticoids. MicroRNAs (miR) may play a role in the wasting process. Activation of the forkhead box O3 (FoxO3) transcription factor causes skeletal muscle atrophy in patients, animals, and cultured cells by increasing the expression of components of the ubiquitin-proteasome and autophagy-lysosome proteolytic systems. To identify microRNAs that potentially modulate the atrophy process, an in silico target analysis was performed and miR-182 was predicted to target FoxO3 mRNA. Using a combination of immunoblot analysis, quantitative real-time RT-PCR, and FoxO3 3′-UTR luciferase reporter genes, miR-182 was confirmed to regulate FoxO3 expression in C2C12 myotubes. Transfection of miR-182 into muscle cells decreased FoxO3 mRNA 30% and FoxO3 protein 67% (P < 0.05) and also prevented a glucocorticoid-induced upregulation of multiple FoxO3 gene targets including MAFbx/atrogin-1, autophagy-related protein 12 (ATG12), cathepsin L, and microtubule-associated protein light chain 3 (LC3). Treatment of C2C12 myotubes with dexamethasone (Dex) (1 μM, 6 h) to induce muscle atrophy decreased miR-182 expression by 63% (P < 0.05). Similarly, miR-182 was decreased 44% (P < 0.05) in the gastrocnemius muscle of rats injected with streptozotocin to induce diabetes compared with controls. Finally, miR-182 was present in exosomes isolated from the media of C2C12 myotubes and Dex increased its abundance. These data identify miR-182 as an important regulator of FoxO3 expression that participates in the control of atrophy-inducing genes during catabolic diseases. © 2014 the American Physiological Society.

Fowler C.B.,Armed Forces Institute of Pathology | Fowler C.B.,Biomedical Laboratory Research and Development Service | Waybright T.J.,U.S. National Cancer Institute | Veenstra T.D.,U.S. National Cancer Institute | And 2 more authors.
Journal of Proteome Research | Year: 2012

Formaldehyde-fixed, paraffin-embedded (FFPE) tissue repositories represent a valuable resource for the retrospective study of disease progression and response to therapy. However, the proteomic analysis of FFPE tissues has been hampered by formaldehyde-induced protein modifications, which reduce protein extraction efficiency and may lead to protein misidentification. Here, we demonstrate the use of heat augmented with high hydrostatic pressure (40,000 psi) as a novel method for the recovery of intact proteins from FFPE mouse liver. When FFPE mouse liver was extracted using heat and elevated pressure, there was a 4-fold increase in protein extraction efficiency, a 3-fold increase in the extraction of intact proteins, and up to a 30-fold increase in the number of nonredundant proteins identified by mass spectrometry, compared to matched tissue extracted with heat alone. More importantly, the number of nonredundant proteins identified in the FFPE tissue was nearly identical to that of matched fresh-frozen tissue. © This article not subject to U.S. Copyright. Published 2012 by the American Chemical Society.

Fowler C.B.,Armed Forces Institute of Pathology | Fowler C.B.,Biomedical Laboratory Research and Development Service | Chesnick I.E.,Armed Forces Institute of Pathology | Moore C.D.,Armed Forces Institute of Pathology | And 2 more authors.
PLoS ONE | Year: 2010

Background: Proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen eggwhite lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing. Principal Findings: In this study, we demonstrate the utility of elevated hydrostatic pressure as a method for efficient protein recovery from FFPE mouse liver tissue and a complex multi-protein FFPE tissue surrogate comprised of hen eggwhite lysozyme, bovine carbonic anhydrase, bovine ribonuclease A, bovine serum albumin, and equine myoglobin (55:15:15:10:5 wt%). Mass spectrometry of the FFPE tissue surrogates retrieved under elevated pressure showed that both the low and high-abundance proteins were identified with sequence coverage comparable to that of the surrogate mixture prior to formaldehyde treatment. In contrast, non-pressure-extracted tissue surrogate samples yielded few positive and many false peptide identifications. Studies with soluble formalin-treated bovine ribonuclease A demonstrated that pressure modestly inhibited the rate of reversal (hydrolysis) of formaldehyde-induced protein cross-links. Dynamic light scattering studies suggest that elevated hydrostatic pressure and heat facilitate the recovery of proteins free of formaldehyde adducts and cross-links by promoting protein unfolding and hydration with a concomitant reduction in the average size of the protein aggregates. Conclusions: These studies demonstrate that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues in a form suitable for proteomic analysis.

Chesnick I.E.,Armed Forces Institute of Pathology | Mason J.T.,Armed Forces Institute of Pathology | O'Leary T.J.,Biomedical Laboratory Research and Development Service | Fowler C.B.,Armed Forces Institute of Pathology | Fowler C.B.,Biomedical Laboratory Research and Development Service
Journal of Cancer | Year: 2010

Formaldehyde fixation and paraffin-embedding remains the most widely used technique for processing cancer tissue specimens for pathologic examination, the study of tissue morphology, and archival preservation. However, formaldehyde penetration and fixation is a slow process, requiring a minimum of 15 hr for routine processing of pathology samples. Routinely fixed samples often have a well-fixed outer rim, with a poorly-fixed inner core of tissue. In this study, we show that the application of elevated pressure up to 15,000 psi improves the rate of formaldehyde fixation by approximately 5 to 7-fold while preserving the tissue morphology of porcine liver. The tissue also exhibited much more uniform formaldehyde penetration after 30-60 min incubation under elevated pressure than samples fixed for the same length of time at atmospheric pressure. © Ivyspring International Publisher.

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