A reverse phase protein array based phospho-antibody characterization approach and its applicability for clinical derived tissue specimens
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...
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| Published in | Scientific reports Vol. 12; no. 1; pp. 22373 - 13 |
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| Main Authors | , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
London
Nature Publishing Group UK
26.12.2022
Nature Publishing Group Nature Portfolio |
| Subjects | |
| Online Access | Get full text |
| ISSN | 2045-2322 2045-2322 |
| DOI | 10.1038/s41598-022-26715-9 |
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| Abstract | 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. |
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| AbstractList | 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. Abstract 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. 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.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. |
| ArticleNumber | 22373 |
| Author | Lu, Aiping Wang, Nan Song, Zhentao Treumann, Achim Ding, Zhiyong Zhang, Li Sun, Tao Ying, Qi Liu, Zhaojian |
| Author_xml | – sequence: 1 givenname: Nan surname: Wang fullname: Wang, Nan email: nan.wang@fynnbio.com organization: Mills Institute for Personalized Cancer Care, Fynn Biotechnologies – sequence: 2 givenname: Li surname: Zhang fullname: Zhang, Li email: leelazhl@163.com organization: Department of Pathology, Beijing Cancer Hospital – sequence: 3 givenname: Qi surname: Ying fullname: Ying, Qi organization: Mills Institute for Personalized Cancer Care, Fynn Biotechnologies – sequence: 4 givenname: Zhentao surname: Song fullname: Song, Zhentao organization: Mills Institute for Personalized Cancer Care, Fynn Biotechnologies – sequence: 5 givenname: Aiping surname: Lu fullname: Lu, Aiping organization: Department of Pathology, Beijing Cancer Hospital – sequence: 6 givenname: Achim surname: Treumann fullname: Treumann, Achim organization: Newcastle University Protein and Proteome Analysis, Newcastle University, KBI Biopharma BV – sequence: 7 givenname: Zhaojian surname: Liu fullname: Liu, Zhaojian organization: Department of Cell Biology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University – sequence: 8 givenname: Tao surname: Sun fullname: Sun, Tao organization: Department of Haematology, Qilu Hospital, Cheeloo College of Medicine, Shandong University – sequence: 9 givenname: Zhiyong surname: Ding fullname: Ding, Zhiyong organization: Mills Institute for Personalized Cancer Care, Fynn Biotechnologies |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/36572710$$D View this record in MEDLINE/PubMed |
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| CitedBy_id | crossref_primary_10_1016_j_mcpro_2024_100830 crossref_primary_10_1021_acsomega_4c09289 crossref_primary_10_1016_j_sciaf_2024_e02308 |
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| SubjectTerms | 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 |
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| Title | A reverse phase protein array based phospho-antibody characterization approach and its applicability for clinical derived tissue specimens |
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