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Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue
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A well-based reverse-phase protein array of formalin-fixed paraffin-embedded tissue.

Joon-Yong Chung1, Stephen M Hewitt

  • 1Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, MSC 1500, Bethesda, Maryland, MD, 20892, USA.

Methods in Molecular Biology (Clifton, N.J.)
|June 6, 2015
PubMed
Summary

A new method uses reverse-phase protein array (RPPA) to analyze proteins in formalin-fixed paraffin-embedded (FFPE) tissues. This technique overcomes challenges from protein cross-linking, enabling biomarker discovery for disease detection and therapeutics.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Clinical Proteomics

Background:

  • Tissue biomarkers are crucial for disease state definition, early detection, and targeted therapeutics.
  • Formalin-fixed paraffin-embedded (FFPE) tissues are standard for clinical histology but challenging for proteomic analysis due to protein cross-linking.
  • Existing proteomic methods face difficulties analyzing FFPE tissue samples.

Purpose of the Study:

  • To develop a robust methodology for proteomic analysis of FFPE tissues.
  • To overcome the limitations of formalin fixation in protein analysis.
  • To enable biomarker discovery and hypothesis testing using clinical FFPE samples.

Main Methods:

  • Development of a well-based reverse-phase protein array (RPPA) platform.
  • Implementation of a novel electrochemiluminescence detection system.
  • Utilized laser capture dissection for protein isolation from FFPE tissue, achieving high protein yield (29.44 ± 7.8 μg/mm³).

Main Results:

  • Demonstrated robust protein isolation from FFPE tissue.
  • Achieved measurable signal differences for proteins using minimal FFPE samples (as few as 500 laser capture dissection shots).
  • Developed protein lysates stable for up to 2 months at room temperature when dried and vacuum-sealed.

Conclusions:

  • The developed RPPA methodology is effective for proteomic analysis of FFPE tissues.
  • This approach facilitates biomarker discovery and validation in clinical settings.
  • The method provides a direct opportunity to test hypotheses within clinical trials and tissue repositories.