A reverse phase protein array based phospho-antibody characterization approach and its applicability for clinical derived tissue specimens

• Published in: Scientific reports Vol. 12; no. 1; pp. 22373 - 13
• Main Authors: Wang, Nan, Zhang, Li, Ying, Qi, Song, Zhentao, Lu, Aiping, Treumann, Achim, Liu, Zhaojian, Sun, Tao, Ding, Zhiyong
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 26.12.2022; Nature Publishing Group; Nature Portfolio
• Subjects: 631/61; 631/67; 692/4017; 692/4028; Alkaline phosphatase; Antibodies; Cell signaling; Formaldehyde; Humanities and Social Sciences; Humans; Lung cancer; Lung Neoplasms - diagnosis; Lysis; Melanoma; multidisciplinary; Paraffin; Paraffin Embedding - methods; Protein Array Analysis - methods; Protein arrays; Proteins; Proteomics; Reproducibility; Reproducibility of Results; Science; Science (multidisciplinary); Tissue Fixation - methods; Tissues
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-022-26715-9

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. We developed a reverse phase protein array (RPPA) based phosphor-antibody characterization approach by taking advantage of the lysis buffer compatible with alkaline phosphatase (AP) treatment that differs from the conventional RPPA antibody validation procedure and applied it onto fresh frozen (FF) and formalin-fixed and paraffin-embedded tissue (FFPE) to test its applicability. By screening 106 phospho-antibodies using RPPA, we demonstrated that AP treatment could serve as an independent factor to be adopted for rapid phospho-antibody selection. We also showed desirable reproducibility and specificity in clincical specimens indicating its potential for tissue-based phospho-protein profiling. Of further clinical significance, using the same approach, based on melanoma and lung cancer FFPE samples, we showed great interexperimental reproducibility and significant correlation with pathological markers in both tissues generating meaningful data that match clinical features. Our findings set a benchmark of an efficient workflow for phospho-antibody characterization that is compatible with high-plex clinical proteomics in precison oncology.