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Realizing the Promise of Reverse Phase Protein Arrays for Clinical, Translational, and Basic Research: A Workshop Report

The RPPA (Reverse Phase Protein Array) Society
Open AccessPublished:April 28, 2014DOI:https://doi.org/10.1074/mcp.O113.034918
      Reverse phase protein array (RPPA) technology introduced a miniaturized “antigen-down” or “dot-blot” immunoassay suitable for quantifying the relative, semi-quantitative or quantitative (if a well-accepted reference standard exists) abundance of total protein levels and post-translational modifications across a variety of biological samples including cultured cells, tissues, and body fluids. The recent evolution of RPPA combined with more sophisticated sample handling, optical detection, quality control, and better quality affinity reagents provides exquisite sensitivity and high sample throughput at a reasonable cost per sample. This facilitates large-scale multiplex analysis of multiple post-translational markers across samples from in vitro, preclinical, or clinical samples. The technical power of RPPA is stimulating the application and widespread adoption of RPPA methods within academic, clinical, and industrial research laboratories.
      Advances in RPPA technology now offer scientists the opportunity to quantify protein analytes with high precision, sensitivity, throughput, and robustness. As a result, adopters of RPPA technology have recognized critical success factors for useful and maximum exploitation of RPPA technologies, including the following:
      • preservation and optimization of pre-analytical sample quality,
      • application of validated high-affinity and specific antibody (or other protein affinity) detection reagents,
      • dedicated informatics solutions to ensure accurate and robust quantification of protein analytes, and
      • quality-assured procedures and data analysis workflows compatible with application within regulated clinical environments.
      In 2011, 2012, and 2013, the first three Global RPPA workshops were held in the United States, Europe, and Japan, respectively. These workshops provided an opportunity for RPPA laboratories, vendors, and users to share and discuss results, the latest technology platforms, best practices, and future challenges and opportunities. The outcomes of the workshops included a number of key opportunities to advance the RPPA field and provide added benefit to existing and future participants in the RPPA research community. The purpose of this report is to share and disseminate, as a community, current knowledge and future directions of the RPPA technology.
      Reverse phase protein array (RPPA)
      The abbreviations used are: RPPA, reverse phase protein array; CLIA, Clinical Laboratory Improvement Amendments; IHC, immunohistochemistry; FDA, U.S. Food and Drug Administration.
      1The abbreviations used are: RPPA, reverse phase protein array; CLIA, Clinical Laboratory Improvement Amendments; IHC, immunohistochemistry; FDA, U.S. Food and Drug Administration.
      technology represents a highly efficient and cost-effective descendent of miniaturized immunoassays that use a sandwich format for antigen capture (
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      Multi-analyte immunoassay.
      ,
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      Multianalyte microspot immunoassay—microanalytical “compact disk” of the future.
      ). Immunoassay arrays were generally sandwich-style assays in which a series of antibodies immobilized on the solid phase were used to capture the analyte of interest (“antibody capture”), with a second antibody, directed against a different epitope on the same protein, used as a detection molecule. In contrast, in “reverse phase” the analytes (antigens) are immobilized on the solid phase (usually nitrocellulose) and subsequently probed with an antibody or other affinity reagent toward a specific target. The term “reverse phase protein microarray” was coined by Paweletz et al. in a paper describing the implementation of the technology for application to cell signaling analysis of laser capture microdissected pre-malignant prostate lesions (
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      Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front.
      ). Since then, terms used in the literature have included “lysate array” (
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      ). For the purposes of this report, and in an attempt to develop a consensus terminology, we use “reverse phase protein array.”
      RPPA technology is dependent on the availability of high-quality monospecific affinity reagents, usually antibodies that can detect with high affinity and specificity a protein or post-translationally modified protein on a solid matrix. Further international efforts such as the Human Protein Atlas Project, Antibodypedia, NCI's Antibody Characterization Program, the Human Antibody Initiative, and aptamerbase are underway to develop, catalog, and validate well-characterized libraries of high-quality affinity reagents that can be used by the community. However, it is important to understand that quality control at each step is paramount for the success of RPPA, in particular in the selection, validation, and implementation of affinity reagents. Challenges associated with this are discussed later in the paper. A number of Web-based resources have recently come online that provide details of antibody validation protocols and published lists of validated RPPA antibodies in current use, including the following:
      • “Antibody Lists and Protocols,” available from The MD Anderson Cancer Center's Functional Proteomics RPPA Core Facility
      • Deutsches Krebsforschungszentrum's page on current protein microarray projects, including RPPA projects
      • A discussion of protein microarray systems from Zeptosens Bioanalytical Solutions
      Furthermore, as the technology is based on a sample-down approach, it is possible to generate and store additional slides (sample arrays) so that further analysis can be performed as new affinity reagents become available or new hypotheses need to be tested. Thus, RPPA provides a highly flexible tool for supporting functional proteomic studies.
      RPPA technology has been applied to a diverse range of sample types to achieve a multiplex quantitative measurement of a large number of analytes extracted from a relatively small number of cells. The technology can be used for protein signal pathway mapping in animal models from Drosophila to mouse, in cell lines and xenografts, and in clinical sample profiling. The biological input can consist of enriched cell populations from tissue microdissection (
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      ), or subcellular fractions.
      Jiyong Liang and Gordon Mills, unpublished.
      2Jiyong Liang and Gordon Mills, unpublished.
      RPPA technology has also been successfully applied to serum/plasma (
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      Screening for C3 deficiency in newborns using microarrays.
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      Validation of reverse phase protein array for practical screening of potential biomarkers in serum and plasma: accurate detection of CA19–9 levels in pancreatic cancer.
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      Core-shell hydrogel particles harvest, concentrate and preserve labile low abundance biomarkers.
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      The heme degradation pathway is a promising serum biomarker source for the early detection of Alzheimer's disease.
      ). The technology is uniquely suited for profiling the state of in vivo signaling networks because of its minimal total cellular volume requirements, high sensitivity (picomole-to-femtomole range), and excellent precision (<15% cv) (
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      Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front.
      ,
      • Tibes R.
      • Qiu Y.
      • Lu Y.
      • Hennessy B.
      • Andreeff M.
      • Mills G.B.
      • Kornblau S.M.
      Reverse phase protein array: validation of a novel proteomic technology and utility for analysis of primary leukemia specimens and hematopoietic stem cells.
      ,
      • VanMeter A.J.
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      Laser capture microdissection and protein microarray analysis of human non-small cell lung cancer: differential epidermal growth factor receptor (EGPR) phosphorylation events associated with mutated EGFR compared with wild type.
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      NormaCurve: a SuperCurve-based method that simultaneously quantifies and normalizes reverse phase protein array data.
      ). Reverse phase protein arrays allow quantitative analysis of phosphorylated, glycosylated, acetylated, cleaved, or total cellular proteins from multiple samples as long as specific detection reagents of high quality are available (
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      ). The dot blot approach, which is dependent on the detection of a single epitope by an affinity reagent, usually an antibody, is particularly applicable to clinical samples, as it is less sensitive to protein quality than is a sandwich antibody-like approach in which two independent epitopes and the intervening region must be intact for quantitative analysis. Indeed, with a number of caveats, RPPA can be applied to at least a subset of targets from formalin-fixed paraffin-embedded patient samples (
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      Molecular analysis of HER2 signaling in human breast cancer by functional protein pathway activation mapping.
      ).
      The RPPA format has been successfully implemented in a variety of formats by a large number of international laboratories. Each laboratory has made significant technical improvements at many stages or has adapted the technology for a particular new use. For example, improvements have been reported concerning the substratum and data capture. Functionalized glass (
      • Seurynck-Servoss S.L.
      • White A.M.
      • Baird C.L.
      • Rodland K.D.
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      Evaluation of surface chemistries for antibody microarrays.
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      ), hydrogel (
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      Antibody microarray profiling of human prostate cancer sera: antibody screening and identification of potential biomarkers.
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      • Bally M.
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      Multilayers of hydrogels loaded with microparticles: a fast and simple approach for microarray manufacturing.
      ), PVDF (
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      Immobilon-P transfer membrane: applications and utility in protein biochemical analysis.
      ,
      • Dalessio J.
      • Ashley R.
      Highly sensitive enhanced chemiluminescence immunodetection method for herpes simplex virus type 2 Western immunoblot.
      ), macroporous silicon (
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      • Guanti G.
      • Simone C.
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      Porous silicon surfaces: a candidate substrate for reverse protein arrays in cancer biomarker detection.
      ), nitrocellulose polymers (
      • Stillman B.A.
      • Tonkinson J.L.
      FAST slides: a novel surface for microarrays.
      ,
      • Tonkinson J.L.
      • Stillman B.A.
      Nitrocellulose: a tried and true polymer finds utility as a post-genomic substrate.
      ) (Grace Bio-Labs; Maine Manufacturing; Sartorius), and planar wave guide surfaces (ZEPTOSENS) (
      • Pawlak M.
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      • Schneider M.J.
      • Oroszlan P.
      • Ehrat M.
      Zeptosens' protein microarrays: a novel high performance microarray platform for low abundance protein analysis.
      ) have all been successfully implemented to improve sensitivity, spot morphology, precision, and accuracy. Further marked improvements have been made in informatics approaches to deal with sample handling, regional staining correction, quality control, and the identification of high-quality samples and reagents. In many cases these have been integrated into publicly available algorithms such as “Supercurve” (
      • Hu J.
      • He X.
      • Baggerly K.A.
      • Coombes K.R.
      • Hennessy B.T.
      • Mills G.B.
      Non-parametric quantification of protein lysate arrays.
      ), “Normacurve” (
      • Troncale S.
      • Barbet A.
      • Coulibaly L.
      • Henry E.
      • He B.
      • Barillot E.
      • Dubois T.
      • Hupé P.
      • de Koning L.
      NormaCurve: a SuperCurve-based method that simultaneously quantifies and normalizes reverse phase protein array data.
      ), and the RPPanalyzer that is available as an R-Package on the CRAN platform (
      • Mannsperger H.A.
      • Gade S.
      • Henjes F.
      • Beissbarth T.
      • Korf U.
      RPPanalyzer: analysis of reverse-phase protein array data.
      ).
      The technology has entered the biotechnology sector under two models: (i) a fee-for-service model, and (ii) as a research tool used in basic and clinical research. Recently, RPPA technology graduated to use in national clinical trials (
      • Mueller C.
      • Liotta L.A.
      • Espina V.
      Reverse phase protein microarrays advance to use in clinical trials.
      ) in a focused personalized medicine trial, and it has become an integral part of large-scale cell line and patient sample characterization efforts such as the Cancer Cell Line Encyclopedia, the Cancer Genome Atlas, and the ISPY2 adaptive design clinical trial.
      The first three Global RPPA Workshops, held in Houston, TX (Oct. 10–11, 2011), Edinburgh, UK (Nov. 12–13, 2012), and Kobe, Japan (Nov. 12–13, 2013), highlighted that, at that point in time, it seemed particularly appropriate to gather together the collective wisdom of the RPPA user community for several important goals. The first goal was to share and disseminate the latest technologies, algorithms, databases, and best practices from each of the participating labs. The second goal was to share lists of validated antibodies and other affinity reagents applicable to RPPA to create a common database for all RPPA users. As the validation of high-quality affinity reagents remains the rate-limiting factor for the implementation of RPPA, a community effort would increase utility, decrease redundancy, and decrease costs. A third goal was the development of general recommendations for sample collection and handling, arraying, data collection, and bioinformatics. The institution of standardized protocols was not the goal of the working group, as the technology is still evolving and a need for flexibility and innovation remains paramount. Nevertheless, the establishment of guiding principles and sharing of best practices and algorithms for quality control for RPPA technology can be essential for (a) accelerating the learning curve for new users, (b) increasing the utility of the platform, (c) clinical trial design and approval, and (d) use of the technology under College of Academic Pathologists/CLIA compliance. The creation of an RPPA society and the organization of yearly workshops were considered essential in order for these goals to be attained. Here, we aim to share and disseminate, as a community, current knowledge and future directions of the RPPA technology. These will now be discussed in further detail.

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