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 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 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.

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 Extraction and Analysis of Diagnostically Useful Proteins from Formalin-fixed, Paraffin-embedded Tissue Sections

1998 ◽  
Vol 46 (3) ◽  
pp. 397-403 ◽  
Author(s):  
Kimimasa Ikeda ◽  
Takushi Monden ◽  
Toshiyuki Kanoh ◽  
Masaki Tsujie ◽  
Hikaru Izawa ◽  
...  
Keyword(s):  
Western Blot ◽  
Cyclin D1 ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Protein Extraction ◽  
Simple Method ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

We describe and discuss a method of protein extraction for Western blot analysis from formalin-fixed, paraffin-embedded tissue sections. From 5-mm2 50-μm-thick tissue sections, an abundance of proteins could be extracted by incubating the sections in lysis buffer containing 2% sodium dodecyl sulfate (SDS) at 100C for 20 min followed by incubation at 60C for 2 hr. Extracts yielded discernible protein bands ranging from 10 kD to 120 kD as identified by SDS-polyacrylamide gel electrophoresis (PAGE). Western blot analysis successfully detected membrane-bound protein such as E-cadherin, cytosolic protein such as β-catenin, and nuclear proteins including proliferating cell nuclear antigen (PCNA), mutant-type p53, cyclin D1, cyclin E, and cyclin-dependent kinases (CDKs). With this technique, we could examine cyclin D1 and CDK2 expression in small adenomas compared with cancer tissues and normal mucosa. The simple method of protein extraction described here should make it possible to use large-scale archives of formalin-fixed, paraffin-embedded samples for Western blot analysis, and its application could lead to detailed analysis of protein expression. This new technique should yield valuable information for molecular biology.

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).

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