Preparation and Use of Reverse Protein Microarrays

Abstract

Reverse-phase protein array (RPPA) is a multiplex, high-throughput proteomic technique for profiling the activation status of signal transduction pathways involved in cancer survival and progression, potentially allowing for identification of new biomarkers and drug targets. On RPPA, the entire patient proteome is immobilized on a spot and single proteins can be quantified across a set of samples, spotted on the same array, with high specificity and sensitivity. Array immunostaining and signal amplification systems are used to generate a signal proportional to the concentration of the analyte. Dedicated scanners and software are used to detect spots, measure intensity, subtract background, normalize signal, and generate a numeric value as output. The generated output file is then analyzed using several different bioinformatic and biostatistical tools. In this unit, the RPPA procedure is described in depth, from sample handling and preparation to data analysis, with particular emphasis on tissue sample analysis.

Enrichment of PI3K-AKT–mTOR Pathway Activation in Hepatic Metastases from Breast Cancer

“…Microdissected cells were lysed in a 1:1 mixture of T-PER (Tissue Protein Extraction Reagent; Pierce) and 2× Tris-glycine SDS sample buffer (Invitrogen) containing 5% 2-mercaptoethanol as previously described, and boiled for 8 minutes before printing arrays (34). …”

“…Signal detection was achieved using a biotinylated goat antirabbit secondary antibody (1:7,500 and 1:5,000 for the discovery and validation set, respectively; Vector Laboratories) coupled with a biotinyl-tyramide–based amplification system (34). The IRDye 680RD Streptavidin (LI-COR Biosciences; dilution 1:50 in PBS supplemented with 1% BSA) or the Cy5 Strepatavidin (KPL; dilution 1:100 in PBS supplemented with 1% BSA) fluorescent detection systems were used for the discovery and validation set, respectively.…”

“…The IRDye 680RD Streptavidin (LI-COR Biosciences; dilution 1:50 in PBS supplemented with 1% BSA) or the Cy5 Strepatavidin (KPL; dilution 1:100 in PBS supplemented with 1% BSA) fluorescent detection systems were used for the discovery and validation set, respectively. Finally, to quantify the amount of protein in each sample, selected slides were stained with Sypro Ruby Protein Blot Stain (Molecular Probes) following manufacturer’s recommendation (34). …”

Reverse Phase Protein Arrays: Mapping the Path Towards Personalized Medicine

“…Despite the algorithm selected for data reduction, the typical data analysis workflow follows the same general processes: a) Determine spot intensity values (pixel intensity/fluorescence intensity) [45, 46] and local spot area background; b) Select data analysis methods (e.g. MicroVigene [47], RPPA Analysis Suite [48], NormaCurve/SuperCurve [49], RPPApipe [50]; modified PSCAN Dose Interpolation Algorithm (DI 25 ) [51], RPPAnalyzer Toolbox [52]), c) Annotate data; d) Calculate differential expression; e) Generate graphical representations of the data; and f) Perform bioinformatics such as unsupervised two-way hierarchical clustering to identify clusters of samples and endpoints.…”

A pilot study exploring the molecular architecture of the tumor microenvironment in human prostate cancer using laser capture microdissection and reverse phase protein microarray

“…For the purpose of this work stroma cells were captured if they met the following criteria: 1) the stroma was surrounded by epithelium on all four corners of Arcturus microscope field at a magnification of 20×; 2) the stroma was in direct contact with the external edge of the tumor (distance between tumor and stroma was ≤50 μm); 3) when present, lymphocyte agglomerates were not captured. Microdissected material was stored at −80 °C and cell lysates were prepared for RPPA as previously described (Pin et al., 2014; Baldelli et al., 2015a).…”