Reverse Phase Protein Arrays (RPPA) represent a very promising sensitive and precise high-throughput technology for the quantitative measurement of hundreds of signaling proteins in biological and clinical samples. This array format allows quantification of one protein or phosphoprotein in multiple samples under the same experimental conditions at the same time. Moreover, it is suited for signal transduction profiling of small numbers of cultured cells or cells isolated from human biopsies, including formalin fixed and paraffin embedded (FFPE) tissues. Owing to the much easier sample preparation, as compared to mass spectrometry based technologies, and the extraordinary sensitivity for the detection of low-abundance signaling proteins over a large linear range, RPPA have the potential for characterization of deregulated interconnecting protein pathways and networks in limited amounts of sample material in clinical routine settings. Current aspects of RPPA technology, including dilution curves, spotting, controls, signal detection, antibody validation, and calculation of protein levels are addressed.

Differences in protein levels and the activation of signaling pathways have been extensively studied in tumor tissues, but the implementation of protein profiling methods in routine hospital workflows lags far behind that of nucleic acid-based approaches. In this review, major technologies that are currently used for measuring protein abundances in human tissues are highlighted, and for each method several examples are provided. We differentiate between extract-based and section-based methods that are each further divided into targeted- and discovery-based approaches (i.e., when the proteins to be analyzed are known or for finding promising new biomarker candidates, respectively). Current problems in protein profiling are addressed and ways in which protein profiling can successfully be implemented in routine clinical workflows are shown.