scholarly journals Optimization of Urea Based Protein Extraction from Formalin-Fixed Paraffin-Embedded Tissue for Shotgun Proteomics

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Stephen A. Luebker ◽  
Scott A. Koepsell
Keyword(s):  
Proteomic Analysis ◽  
Protein Extraction ◽  
Protein Detection ◽  
Ffpe Tissue ◽  
Extraction Temperature ◽  
Esi Ms ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Buffer Composition ◽  
Formalin Fixed

Urea based protein extraction of formalin-fixed paraffin-embedded (FFPE) tissue provides the most efficient workflow for proteomics due to its compatibility with liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). This study optimizes the use of urea for proteomic analysis of clinical FFPE tissue. A series of protein extraction conditions manipulating temperature and buffer composition were compared to reduce carbamylation introduced by urea and increase protein detection. Each extraction was performed on a randomized pair of serial sections of homogenous FFPE tissue and analyzed with LC-ESI-MS/MS. Results were compared in terms of yield, missed cleavages, and peptide carbamylation. Lowering extraction temperature to 60°C decreased carbamylation at the cost of decreased protein detection and yield. Protein extraction for at least 20 minutes at 95°C followed by 60°C for 2 hours maximized total protein yield while maintaining protein detection and reducing carbamylation by 7.9%. When accounting for carbamylation during analysis, this modified extraction temperature provides equivalent peptide and protein detection relative to the commercially available Qproteome® FFPE Tissue Kit. No changes to buffer composition containing 7 M urea, 2 M thiourea, and 1 M ammonium bicarbonate resulted in improvements to control conditions. Optimized urea in-solution digestion provides an efficient workflow with maximized yields for proteomic analysis of clinically relevant FFPE tissue.

scholarly journals HYPERsol: flash-frozen results from archival FFPE tissue for clinical proteomics

2019 ◽  
Author(s):  
Dylan M. Marchione ◽  
Ilyana Ilieva ◽  
Benjamin A. Garcia ◽  
Darryl J. Pappin ◽  
John P. Wilson ◽  
...  
Keyword(s):  
Protein Extraction ◽  
Ffpe Tissue ◽  
Clinical Proteomics ◽  
High Yield ◽  
Proteome Coverage ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Proteomics Research ◽  
Formalin Fixed

Massive formalin-fixed, paraffin-embedded (FFPE) tissue archives exist worldwide, representing a potential gold mine for clinical proteomics research. However, current protocols for FFPE proteomics lack standardization, efficiency, reproducibility, and scalability. Here we present High-Yield Protein Extraction and Recovery by direct SOLubilization (HYPERsol), an optimized workflow using adaptive-focused acoustics (AFA) ultrasonication and S-Trap sample processing that enables proteome coverage and quantification from FFPE samples comparable to that achieved from flash-frozen tissue (average R = 0.936).

scholarly journals Comparison of multiple protein extraction buffers for GeLC-MS/MS proteomic analysis of liver and colon formalin-fixed, paraffin-embedded tissues

2016 ◽  
Vol 12 (2) ◽  
pp. 553-565 ◽  
Author(s):  
Valérie Broeckx ◽  
Kurt Boonen ◽  
Lentel Pringels ◽  
Xavier Sagaert ◽  
Hans Prenen ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Protein Extraction ◽  
Multiple Protein ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Comparison of protein extraction buffers and overall variation in formalin-fixed paraffin-embedded tissue using the same proteomic workflow.

scholarly journals Protein Extraction of Formalin-fixed, Paraffin-embedded Tissue Enables Robust Proteomic Profiles by Mass Spectrometry

2009 ◽  
Vol 57 (9) ◽  
pp. 849-860 ◽  
Author(s):  
Marshall S. Scicchitano ◽  
Deidre A. Dalmas ◽  
Rogely W. Boyce ◽  
Heath C. Thomas ◽  
Kendall S. Frazier
Keyword(s):  
Mass Spectrometry ◽  
Proteomic Analysis ◽  
Protein Quality ◽  
Protein Extraction ◽  
Cutting Temperature ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Optimal Cutting Temperature ◽  
Formalin Fixed

Global mass spectrometry (MS) profiling and spectral count quantitation are used to identify unique or differentially expressed proteins and can help identify potential biomarkers. MS has rarely been conducted in retrospective studies, because historically, available samples for protein analyses were limited to formalin-fixed, paraffin-embedded (FFPE) archived tissue specimens. Reliable methods for obtaining proteomic profiles from FFPE samples are needed. Proteomic analysis of these samples has been confounded by formalin-induced protein cross-linking. The performance of extracted proteins in a liquid chromatography tandem MS format from FFPE samples and extracts from whole and laser capture microdissected (LCM) FFPE and frozen/optimal cutting temperature (OCT)- embedded matched control rat liver samples were compared. Extracts from FFPE and frozen/OCT-embedded livers from atorvastatin-treated rats were further compared to assess the performance of FFPE samples in identifying atorvastatin-regulated proteins. Comparable molecular mass representation was found in extracts from FFPE and OCT-frozen tissue sections, whereas protein yields were slightly less for the FFPE sample. The numbers of shared proteins identified indicated that robust proteomic representation from FFPE tissue and LCM did not negatively affect the number of identified proteins from either OCT-frozen or FFPE samples. Subcellular representation in FFPE samples was similar to OCT-frozen, with predominantly cytoplasmic proteins identified. Biologically relevant protein changes were detected in atorvastatin-treated FFPE liver samples, and selected atorvastatin-related proteins identified by MS were confirmed by Western blot analysis. These findings demonstrate that formalin fixation, paraffin processing, and LCM do not negatively impact protein quality and quantity as determined by MS and that FFPE samples are amenable to global proteomic analysis.

scholarly journals The Dutch National TissueArchive Portal enables efficient, consistent, and transparent procurement of diagnostic tissue samples for scientific use

2021 ◽  
Author(s):  
Robin Verjans ◽  
Annette H. Bruggink ◽  
Robby Kibbelaar ◽  
Jos Bart ◽  
Aletta Debernardi ◽  
...  
Keyword(s):  
The Netherlands ◽  
Tissue Sample ◽  
Ffpe Tissue ◽  
Tissue Samples ◽  
Internet Portal ◽  
Formalin Fixed Paraffin ◽  
Practical Support ◽  
Formalin Fixed Paraffin Embedded ◽  
The Rich ◽  
Formalin Fixed

AbstractBiobanks play a crucial role in enabling biomedical research by facilitating scientific use of valuable human biomaterials. The PALGA foundation—a nationwide network and registry of histo- and cytopathology in the Netherlands—was established to promote the provision of data within and between pathology departments, and to make the resulting knowledge available for healthcare. Apart from the pathology data, we aimed to utilize PALGA’s nationwide network to find and access the rich wealth of Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples for scientific use.  We implemented the Dutch National TissueArchive Portal (DNTP) to utilize PALGA’s nationwide network for requesting FFPE tissue samples. The DNTP consists of (1) a centrally organized internet portal to improve the assessing, processing, harmonization, and monitoring of the procurement process, while (2) dedicated HUB-employees provide practical support at peripheral pathology departments. Since incorporation of the DNTP, both the number of filed requests for FFPE tissue samples and the amount of HUB-mediated support increased 55 and 29% respectively. In line, the sample procurement duration time decreased significantly (− 47%). These findings indicate that implementation of the DNTP improved the frequency, efficiency, and transparency of FFPE tissue sample procurement for research in the Netherlands. To conclude, the need for biological resources is growing persistently to enable precision medicine. Here, we access PALGA’s national, pathology network by implementation of the DNTP to allow for efficient, consistent, and transparent exchange of FFPE tissue samples for research across the Netherlands.

scholarly journals Discrimination of Aspergillosis, Mucormycosis, Fusariosis, and Scedosporiosis in Formalin-Fixed Paraffin-Embedded Tissue Specimens by Use of Multiple Real-Time Quantitative PCR Assays

2016 ◽  
Vol 54 (11) ◽  
pp. 2798-2803 ◽  
Author(s):  
Elham Salehi ◽  
Mohammad T. Hedayati ◽  
Jan Zoll ◽  
Haleh Rafati ◽  
Maryam Ghasemi ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Aspergillus Flavus ◽  
Ffpe Tissue ◽  
Content Type ◽  
Real Time Quantitative Pcr ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Tissue Specimens ◽  
Formalin Fixed

In a retrospective multicenter study, 102 formalin-fixed paraffin-embedded (FFPE) tissue specimens with histopathology results were tested. Two 4- to 5-μm FFPE tissue sections from each specimen were digested with proteinase K, followed by automated nucleic acid extraction. Multiple real-time quantitative PCR (qPCR) assays targeting the internal transcribed spacer 2 (ITS2) region of ribosomal DNA, using fluorescently labeled primers, was performed to identify clinically important genera and species of Aspergillus , Fusarium , Scedosporium , and the Mucormycetes . The molecular identification was correlated with results from histological examination. One of the main findings of our study was the high sensitivity of the automated DNA extraction method, which was estimated to be 94%. The qPCR procedure that was evaluated identified a range of fungal genera/species, including Aspergillus fumigatus , Aspergillus flavus , Aspergillus terreus , Aspergillus niger , Fusarium oxysporum , Fusarium solani , Scedosporium apiospermum , Rhizopus oryzae , Rhizopus microsporus , Mucor spp., and Syncephalastrum . Fusarium oxysporum and F. solani DNA was amplified from five specimens from patients initially diagnosed by histopathology as having aspergillosis. Aspergillus flavus , S. apiospermum , and Syncephalastrum were detected from histopathological mucormycosis samples. In addition, examination of four samples from patients suspected of having concomitant aspergillosis and mucormycosis infections resulted in the identification of two A. flavus isolates, one Mucor isolate, and only one sample having both R. oryzae and A. flavus . Our results indicate that histopathological features of molds may be easily confused in tissue sections. The qPCR assay used in this study is a reliable tool for the rapid and accurate identification of fungal pathogens to the genus and species levels directly from FFPE tissues.

Impact of routinely employed procedures for tissue processing on the proteomic analysis of formalin-fixed paraffin-embedded tissue

2014 ◽  
Vol 8 (9-10) ◽  
pp. 796-804 ◽  
Author(s):  
Peter Bronsert ◽  
Juliane Weißer ◽  
Martin L. Biniossek ◽  
Markus Kuehs ◽  
Bettina Mayer ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Tissue Processing ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

scholarly journals Quantitative Proteomic Analysis of Formalin Fixed Paraffin Embedded Oral HPV Lesions from HIV Patients

2008 ◽  
Vol 1 (1) ◽  
pp. 40-45 ◽  
Author(s):  
Mohit Raja Jain ◽  
Tong Liu ◽  
Jun Hu ◽  
Marlene Darfler ◽  
Valerie Fitzhugh ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Hiv Patients ◽  
Quantitative Proteomic Analysis ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Oral Hpv ◽  
Formalin Fixed

scholarly journals Incorporation of Cervista Human Papillomavirus 16/18 Assay Into Algorithms for Classifying Human Papillomavirus Status in Formalin-Fixed, Paraffin-Embedded Head and Neck Squamous Carcinoma Specimens

2018 ◽  
Vol 143 (3) ◽  
pp. 356-361
Author(s):  
Ming Guo ◽  
Abha Khanna ◽  
Jianping Wang ◽  
Michelle D. Williams ◽  
Neda Kalhor ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Human Papillomavirus ◽  
Ffpe Tissue ◽  
Hpv 16 ◽  
Hpv Dna ◽  
Formalin Fixed Paraffin ◽  
Hpv Status ◽  
Formalin Fixed Paraffin Embedded ◽  
Hpv Assays ◽  
Formalin Fixed

Context.— Human papillomavirus (HPV) DNA in situ hybridization (ISH) assay and p16 immunohistochemistry (IHC) are used to determine high-risk HPV status in formalin-fixed, paraffin-embedded (FFPE) tissues in oropharyngeal squamous cell carcinoma (SCC). Although high sensitivity and specificity for HPV can be obtained by combined p16 IHC and HPV DNA ISH, the occasional discrepancy between these assays has prompted evaluation of Cervista HPV assays in FFPE tissue from patients with oropharyngeal SCC. Objective.— To compare the efficacy of Cervista HPV 16/18 and Cervista HPV HR assay to that of HPV DNA ISH assay and p16 IHC in FFPE tissue in head and neck squamous cell carcinoma of oropharyngeal origin. Design.— Archived FFPE tissue from 84 patients with SCC of oropharyngeal origin and available HPV DNA ISH and p16 IHC test results were tested with the Cervista HPV 16/18 assay and further verified by polymerase chain reaction (PCR)–based HPV16/18 genotyping tests in cases with discrepancy. Results.— Of the 84 specimens, 75% (63 of 84) were positive and 16% (13 of 84) had discrepant or equivocal findings by p16 IHC and HPV DNA ISH testing. Use of Cervista HPV assays, either to clarify discrepant/equivocal findings or as confirmation after initial p16 IHC/HPV DNA ISH tests, identified 81% (68 of 84) of HPV-positive cases without equivocal HPV results. Five of 13 cases with discrepancy or equivocal HPV DNA ISH results tested positive for HPV16 or HPV18 by Cervista HPV 16/18 assay, which was further confirmed by PCR-based HPV 16/18 genotyping. Conclusions.— The Cervista HPV assays are a reasonable alternative to HPV DNA ISH in determining HPV status in FFPE tissue specimens from patients with oropharyngeal SCC.

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