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Formalin-fixed paraffin-embedded (FFPE) proteome analysis using gel-free and gel-based proteomics.
J. Proteome Res. 9, 4710-4720 (2010)
Formalin-fixed paraffin-embedded (FFPE) tissue has recently gained interest as an alternative to fresh/frozen tissue for retrospective protein biomarker discovery. However, during the fixation process, proteins undergo degradation and cross-linking, making conventional protein analysis technologies problematic. In this study, we have compared several extraction and separation methods for the analysis of proteins in FFPE tissues. Incubation of tissue sections at high temperature with a novel extraction buffer (20 mM Tris-HCl, pH 8.8, 2% SDS, 1% beta-octylglucoside, 200 mM DTT, 200 mM glycine, and a mixture of protease inhibitors) resulted in improved protein recovery. Protein separation by 1-DE followed by LC-ESI MS/MS analysis was the most effective approach to identify proteins, based on the number of peptides reliably identified. Interestingly, a number of peptides were identified in regions of the 1DE not corresponding to their native molecular weights. This is an indication of the formation of protein-protein complexes by cross-linking, and of protein fragmentation due to prolonged sample storage. This study will facilitate the development of future proteomic analysis of FFPE tissue and provide a tool for the validation in archival samples of biomarkers of exposure, prognosis and disease.
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Publication type
Article: Journal article
Document type
Scientific Article
Keywords
Formalin-fixed paraffin-embedded (FFPE); Gel-based; Gel-free; Proteomics; Protein extraction; Peptide modification; Cross-linking
Language
english
Publication Year
2010
HGF-reported in Year
2010
ISSN (print) / ISBN
1535-3893
e-ISSN
1535-3907
Journal
Journal of Proteome Research
Quellenangaben
Volume: 9,
Issue: 9,
Pages: 4710-4720
Publisher
American Chemical Society (ACS)
Reviewing status
Peer reviewed
Institute(s)
Institute of Radiation Biology (ISB)
Institute of Pathology (PATH)
CF Metabolomics & Proteomics (CF-MPC)
Institute of Pathology (PATH)
CF Metabolomics & Proteomics (CF-MPC)
POF-Topic(s)
30202 - Environmental Health
30504 - Mechanisms of Genetic and Environmental Influences on Health and Disease
30203 - Molecular Targets and Therapies
30504 - Mechanisms of Genetic and Environmental Influences on Health and Disease
30203 - Molecular Targets and Therapies
Research field(s)
Radiation Sciences
Enabling and Novel Technologies
Enabling and Novel Technologies
PSP Element(s)
G-500200-001
G-500300-001
G-505700-001
G-500300-001
G-505700-001
PubMed ID
20604508
Scopus ID
77956327955
Erfassungsdatum
2010-10-08