Abstract
Mastering the systematic analysis of tumor tissues on a large scale has long been a technical challenge for proteomics. In 2001, reverse phase protein arrays (RPPA) were added to the repertoire of existing immunoassays, which, for the first time, allowed a profiling of minute amounts of tumor lysates even after microdissection. A characteristic feature of RPPA is its outstanding sample capacity permitting the analysis of thousands of samples in parallel as a routine task. Until today, the RPPA approach has matured to a robust and highly sensitive high-throughput platform, which is ideally suited for biomarker discovery. Concomitant with technical advancements, new bioinformatic tools were developed for data normalization and data analysis as outlined in detail in this review. Furthermore, biomarker signatures obtained by different RPPA screens were compared with another or with that obtained by other proteomic formats, if possible. Options for overcoming the downside of RPPA, which is the need to steadily validate new antibody batches, will be discussed. Finally, a debate on using RPPA to advance personalized medicine will conclude this article.
Highlights
Introduction of a commonly acceptedreverse phase protein arrays (RPPA) data standard would be of high importance in regard to studies dealing with cross-platform data analysis
The x-axis presents the log2 gene expression level and the y-axis reflects log2 fold-change with respect to a reference sample. This concept was employed by the Variable Slope (VS) approach and showed promising results with respect to loading effect correction and variance stabilization, and resulted in RPPA data that showed a good correlation with IHC/fluorescence in situ hybridization (FISH) data available for the same set of samples
Evaluation of this automated approach showed prediction accuracies of 84%–87% when compared to a combined evaluation of three RPPA experts, considering the fact of missing a gold standard of RPPA quality control
Summary
With the development of personalized therapeutics for oncology, the systematic and targeted analysis of selected proteins in tumor tissues is currently receiving increasing interest. Screening clinical tissues for target proteins of potential pharmaceutical interest requires large numbers of well-documented clinical samples to yield statistically relevant data. High capacity platforms such as reverse phase protein arrays (RPPA), for example, are well suited for this purpose. Fundamental changes of the cellular proteome occur immediately post-excision, a process described as cold ischemia. Biochemical processes occurring post-excision need to be taken into account as an important pre-analytical factor that will influence the resulting sample quality and require standardized procedures regarding tissue handling as well as sample preparation to ensure comparable sample quality. To set-up a bio-bank, clinical tissues need to be processed under consistent conditions over many years to exclude artifacts resulting from tissue handling and storage
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