TY - JOUR
T1 - Improved protein extraction and protein identification from archival formalin-fixed paraffin-embedded human aortas
AU - Fu, Zongming
AU - Yan, Kun
AU - Rosenberg, Avraham
AU - Jin, Zhicheng
AU - Crain, Barbara
AU - Athas, Grace
AU - Heide, Richard S.Vander
AU - Howard, Timothy
AU - Everett, Allen D.
AU - Herrington, David
AU - Van Eyk, Jennifer E.
PY - 2013/4
Y1 - 2013/4
N2 - Purpose: Evaluate combination of heat and elevated pressure to enhance protein extraction and quality of formalin-fixed (FF), and FF paraffin-embedded (FFPE) aorta for proteomics. Experiment design: Proteins were extracted from fresh frozen aorta at room temperature (RT). FF and FFPE aortas (3 months and 15 years) were extracted at RT, heat alone, or a combination of heat and high pressure. Protein yields were compared, and digested peptides from the extracts were analyzed with MS. Results: Combined heat and elevated pressure increased protein yield from human FF or FFPE aorta compared to matched tissues with heat alone (1.5-fold) or at RT (8.3-fold), resulting in more proteins identified and with more sequence coverage. The length of storage did adversely affect the quality of proteins from FF tissue. For long-term storage, aorta was preserved better with FFPE than FF alone. Periostin and MGF-E8 were demonstrated suitable for MRM assays from FFPE aorta. Conclusions and clinical relevance: Combination of heat and high pressure is an effective method to extract proteins from FFPE aorta for downstream proteomics. This method opens the possibility for use of archival and often rare FFPE aortas and possibly other tissues available to proteomics for biomarker discovery and quantification.
AB - Purpose: Evaluate combination of heat and elevated pressure to enhance protein extraction and quality of formalin-fixed (FF), and FF paraffin-embedded (FFPE) aorta for proteomics. Experiment design: Proteins were extracted from fresh frozen aorta at room temperature (RT). FF and FFPE aortas (3 months and 15 years) were extracted at RT, heat alone, or a combination of heat and high pressure. Protein yields were compared, and digested peptides from the extracts were analyzed with MS. Results: Combined heat and elevated pressure increased protein yield from human FF or FFPE aorta compared to matched tissues with heat alone (1.5-fold) or at RT (8.3-fold), resulting in more proteins identified and with more sequence coverage. The length of storage did adversely affect the quality of proteins from FF tissue. For long-term storage, aorta was preserved better with FFPE than FF alone. Periostin and MGF-E8 were demonstrated suitable for MRM assays from FFPE aorta. Conclusions and clinical relevance: Combination of heat and high pressure is an effective method to extract proteins from FFPE aorta for downstream proteomics. This method opens the possibility for use of archival and often rare FFPE aortas and possibly other tissues available to proteomics for biomarker discovery and quantification.
KW - Aorta
KW - Formalin-fixed paraffin-embedded
KW - Heat and high-pressure protein extraction
KW - MS
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U2 - 10.1002/prca.201200064
DO - 10.1002/prca.201200064
M3 - Article
C2 - 23339088
AN - SCOPUS:84876333265
SN - 1862-8346
VL - 7
SP - 217
EP - 224
JO - Proteomics - Clinical Applications
JF - Proteomics - Clinical Applications
IS - 3-4
ER -