Reproducibility and Crossplatform Validation of Reverse-Phase Protein Array Data

Byron, Adam

doi:10.1007/978-981-32-9755-5_10

Cited by 7 publications

(6 citation statements)

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“…RPPA is a highly sensitive and accurate technology that can assess hundreds of proteins and signaling nodes under the same experimental conditions using a high-throughput assessment method (>1000 samples/slide) [2,4,9]. The technique is highly sensitive for low-abundance signaling proteins and has the potential to characterize deregulated interconnecting protein pathways with limited sample volumes [4,8].…”

Section: Discussionmentioning

confidence: 99%

“…Since samples were obtained from pediatric patients, sample material was limited and a format requiring limited sample was used. Although there are RPPA platforms that require less material, such as the Zeptosens platform [23], we chose to test a standard RPPA because it is easily available and not costprohibitive [9]. Using the alternative method of extreme protein expression assessment, we identified 25 samples from 28 patients (5%) with excessive sums of extremes, an indication of poor sample quality.…”

Section: Discussionmentioning

confidence: 99%

“…There is concern, however, that preanalytical handling variables, i.e. those changes occurring between sample collection and assay processing, can affect the integrity of protein concentrations [9,10]. Previous work has demonstrated that molecular assessments can be affected by leukemia sample quality.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

The effects of sample handling on proteomics assessed by reverse phase protein arrays (RPPA): Functional proteomic profiling in leukemia

Horton

Hoff

Dijk

et al. 2021

Journal of Proteomics

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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The effects of sample handling on proteomics assessed by reverse phase protein arrays (RPPA): Functional proteomic profiling in leukemia

Horton

Hoff

Dijk

et al. 2021

Journal of Proteomics

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“…Robust, high-quality data are crucial for successful RPPA workflows, especially as RPPA pipelines are continuing to be developed for use in clinical settings [54][55][56][57][58] . Thus, there is a growing need for approaches to record and evaluate the reproducibility of different RPPA platform outputs 59 . To control biological and technical variability in this multicenter study, each cell line was cultured at one center and then lysates were distributed to all centers, so that all platforms analyzed exactly the same batch of protein extract.…”

Section: Experimental Designmentioning

confidence: 99%

Multicenter reverse-phase protein array data integration

Koning

Bernhardt

Macleod

et al. 2021

Preprint

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Among the technologies available for protein biomarker discovery and validation, reverse-phase protein array (RPPA) benefits from unequalled sample throughput. Panels of high-quality antibodies enable the quantification by RPPA of protein abundance and posttranslational modifications in biological specimens with high precision and sensitivity. Incorporation of RPPA technology into clinical and drug development pipelines requires robust assays that generate reproducible results across multiple laboratories. We implemented the first international multicenter pilot study to investigate RPPA workflow variability. We characterized the proteomic responses of a series of breast cancer cells to two cancer drugs. This analysis quantified 86,832 sample spots, representing 108 biological samples, arrayed at three independent RPPA platforms. This unique integrated set of data is publicly available as a resource to the proteomic and cancer research communities to catalyse further analysis and investigation. We anticipate that this dataset will form a reference for the comparison of RPPA workflows and reagents, which can be expanded in the future, and will aid the identification of platform-robust treatment-marker antigens in breast cancer cells.

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“…As most laboratories using RPPA technology have developed a customised set-up, there are essentially as many workflows as there are RPPA platforms. Differences between platforms are diverse and can include the type of printer used to create the arrays, sample spotting conditions, slide substrate chemistry, primary and secondary antibodies used for immunostaining and slide scanner optics 27 . Moreover, no standard tools exist to quantify, normalise and quality control RPPA data.…”

mentioning

confidence: 99%

Integrative analysis of multi-platform reverse-phase protein array data for the pharmacodynamic assessment of response to targeted therapies

Byron

Bernhardt

Ouine

et al. 2020

Sci Rep

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Reverse-phase protein array (RPPA) technology uses panels of high-specificity antibodies to measure proteins and protein post-translational modifications in cells and tissues. The approach offers sensitive and precise quantification of large numbers of samples and has thus found applications in the analysis of clinical and pre-clinical samples. For effective integration into drug development and clinical practice, robust assays with consistent results are essential. Leveraging a collaborative RPPA model, we set out to assess the variability between three different RPPA platforms using distinct instrument set-ups and workflows. Employing multiple RPPA-based approaches operated across distinct laboratories, we characterised a range of human breast cancer cells and their protein-level responses to two clinically relevant cancer drugs. We integrated multi-platform RPPA data and used unsupervised learning to identify protein expression and phosphorylation signatures that were not dependent on RPPA platform and analysis workflow. Our findings indicate that proteomic analyses of cancer cell lines using different RPPA platforms can identify concordant profiles of response to pharmacological inhibition, including when using different antibodies to measure the same target antigens. These results highlight the robustness and the reproducibility of RPPA technology and its capacity to identify protein markers of disease or response to therapy.

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Reproducibility and Crossplatform Validation of Reverse-Phase Protein Array Data

Cited by 7 publications

References 100 publications

The effects of sample handling on proteomics assessed by reverse phase protein arrays (RPPA): Functional proteomic profiling in leukemia

The effects of sample handling on proteomics assessed by reverse phase protein arrays (RPPA): Functional proteomic profiling in leukemia

Multicenter reverse-phase protein array data integration

Integrative analysis of multi-platform reverse-phase protein array data for the pharmacodynamic assessment of response to targeted therapies

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