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

Abstract

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.

Virtual Screening and Reality Verification:Elemene Injectable Emulsion acts on the key targets and pathways of Colorectal Adenoma Cancerization

“…It found key biomarkers with prognostic importance in the molecular typing of CRC (39). It was also widely used to evaluate tumor drug treatment effects and the study of a tumor-related drug resistance mechanism (40,41).…”

A preliminary, prospective study of peripheral neuropathy and cognitive function in patients with breast cancer during taxane therapy

“…The RPPA core facility at MD Anderson Cancer Center used a set of 484 predetermined antibodies for each array, and the results were quantified by a set of MD Anderson algorithms. The raw data for protein expression levels was analyzed using standard web-based protocols [26].…”

Multicenter reverse-phase protein array data integration

“…The dataset consists of original RPPA slide scan images and image quantification of 86,832 sample spots derived from 972 arrayed lysates, incorporating independent biological replicate samples, serial lysate dilution series, and technical replicate sample spots, from three independent RPPA platforms. Integrative analysis of a subset of these data enabled the identification of platform-independent protein markers of breast cancer cell response to drug treatment 48 . We report here the raw and processed data from our assessment of multiple RPPA workflows at different research centers.…”

“…1b-d). Some of the following methods are expanded versions of descriptions in our related work 48 . 48 .…”

“…Nonspecific signals were determined by omitting the primary antibody incubation step. The linear fit of the relative fluorescence intensities for the dilution series of each vehicle controltreated sample was determined for each primary antibody, and >99% of signals derived from neat samples (1.5 mg/ml) were within the linear range of detection 48 . Medians of technical replicate sample spots were calculated, background was corrected, and quality control was performed using RPPanalyzer 51 , and data were written to a CSV file (SampleQuantification2_DKFZ.csv) 49 .…”