Abstract
Background: Systematic quantification of phosphoprotein within cell signaling networks in solid tissues remains challenging and precise quantification in large scale samples has great potential for biomarker identification and validation. Methods:We developed a reverse phase protein array (RPPA) based phosphor-antibody characterization workflow by taking advantage of the lysis buffer compatible with alkaline phosphatase (AP) treatment and here termed it as a bottom-up antibody screening that differs from the conventional RPPA antibody validation procedure and applied it onto fresh frozen and formalin-fixed and paraffin-embedded tissue (FFPE) to test its applicability.Results:We tested the feasibility of this method by screening 106 phospho-antibodies with RPPA first followed by western blots on a panel of cell lines and demonstrated that AP treatment could serve as an independent factor that can be adopted for rapid RPPA phospho-antibody selection. We also performed studies on different clinical materials. For fresh frozen (FF) samples, pre-selected highly-specific antibodies showed a desirable data reproducibility and antibody specificity based on AP treatment indicating a potential for fresh tissue-based phospho-protein RPPA profiling. Of further clinical significance, using the same approach, based on two sets of FFPE samples from 63 melanoma and 40 lung cancer patients, we showed great interexperimental reproducibility and significant correlation with pathological markers MelanA for melanoma as well as a panel of lung cancer biomarkers for subtyping (EGFR, Napsin A, p63/p40, TTF1 and CK7) generating meaningful data that match clinical features. Conclusions:Our findings establish a highly efficient approach for phospho-antibody characterization by taking advantage of RPPA whereby the same methodology can be applied for tissue-based proteomics and phosphoproteomics in clinical assay development and application.