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Published on: September 19, 2018
A reverse phase protein array based phospho-antibody characterization approach and its applicability for clinical
Nan Wang1, Li Zhang2, Qi Ying3
1Mills Institute for Personalized Cancer Care, Fynn Biotechnologies, Floor 22, Overseas Chinese Innovation Zone, Gangxing 3rd Rd, High-Tech and Innovation Zone, Jinan, 250100, China. nan.wang@fynnbio.com.
We developed a novel method for quantifying phosphoproteins in tissues using reverse phase protein arrays (RPPA) and alkaline phosphatase (AP) treatment. This approach enables efficient phospho-antibody selection and reproducible tissue-based phospho-protein profiling for precision oncology.
Area of Science:
- Biochemistry
- Molecular Biology
- Proteomics
Background:
- Accurate quantification of phosphoproteins in solid tissues is crucial for understanding cell signaling and identifying biomarkers.
- Existing methods for large-scale phosphoprotein analysis in tissues face challenges in precision and reproducibility.
- Formalin-fixed and paraffin-embedded (FFPE) tissues are valuable clinical resources but pose technical hurdles for proteomic analysis.
Purpose of the Study:
- To develop and validate a novel reverse phase protein array (RPPA) based approach for phosphoprotein quantification in both fresh frozen (FF) and FFPE tissues.
- To establish an efficient workflow for rapid phospho-antibody selection and characterization using alkaline phosphatase (AP) treatment.
- To demonstrate the clinical utility of the developed method for tissue-based phospho-protein profiling and its correlation with pathological markers.
Main Methods:
- Development of a modified RPPA protocol utilizing a lysis buffer compatible with alkaline phosphatase (AP) treatment for antibody validation.
- Application of the developed RPPA approach to screen 106 phospho-antibodies using both FF and FFPE tissue samples.
- Validation of reproducibility and specificity of the method using clinical specimens, including melanoma and lung cancer FFPE samples.
Main Results:
- Alkaline phosphatase (AP) treatment was identified as an independent factor for rapid and efficient phospho-antibody selection.
- The developed RPPA method demonstrated desirable reproducibility and specificity in clinical specimens.
- Analysis of melanoma and lung cancer FFPE samples showed high interexperimental reproducibility and significant correlation with pathological markers, aligning with clinical features.
Conclusions:
- The novel RPPA-based phosphor-antibody characterization approach provides an efficient workflow for phospho-protein profiling in FF and FFPE tissues.
- This method is compatible with high-plex clinical proteomics, offering significant potential for biomarker discovery and validation in precision oncology.
- The findings establish a benchmark for robust and reproducible tissue-based phosphoproteomic analysis in clinical settings.
