Reverse Phase Protein Arrays-Quantitative Assessment of Multiple Biomarkers in Biopsies for Clinical Use

Abstract

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.

Keywords: FFPE; antibody; cancer; diagnostics; personalized medicine; protein; therapy; tissue.

Figure 1

Schematic presentation of forward phase protein array (A) and reverse phase protein array (B). Protein A: Protein of interest. B–G: Proteins of the lysate that are not recognized by the primary antibody. HRP: Horse radish peroxidase.

Figure 2

Workflow of Reverse Phase Protein Array (RPPA) studies.

Figure 3

Antibody validation by Western Blot using different cell lines. The left panel shows an antibody that is suitable for RPPA analysis as it detects one single protein band at the expected molecular weight. The antibody used for the right panel should not be used for RPPA studies as there are numerous bands besides the proposed “specific” band. 1–4: lysates from breast cancer FFPE-tissue; 5–8: lysates from different cell lines; arrows indicate the expected molecular weight.

Figure 4

Basic steps for antibody validation. For detailed explanations of the different steps see text. WB, Western Blot.

Figure 5

RPPA-spotting pattern detected by chemiluminescent detection. Subgrids comprise 6 samples, each in a five-spot serial dilution plus protein extraction buffer as negative control (36 spots). Grids comprise 16 subgrids and 576 spots (Grid 1–3)/12 subgrids and 432 spots (Grid 4). In total, 180 samples can be spotted per slide (2160 spots). Numbers (1–3) indicate three samples spotted in duplicates. * Smaller grid (3/4 size of Grids 1–3).