Reverse-Phase Protein Microarrays

Abstract

Cancer is the consequence of intra- and extracellular signaling network deregulation that derives from alteration of genetic and proteomic cellular homeostasis. Mapping the individual molecular circuitry of a patient's tumor cells is the starting point for rational personalized therapy.While genes and RNA encode information about cellular status, proteins are considered the engine of the cellular machine, as they are the effective elements that drive cellular functions, such as proliferation, migration, differentiation, and apoptosis. Consequently, investigations of the cellular protein network are considered a fundamental tool to understand cellular functions. In the last decades, increasing interest has been focused on the improvement of new technologies for proteomic analysis. In this context, reverse-phase protein microarrays (RPMAs) have been developed to study and analyze posttranslational modifications that are responsible for principal cell functions and activities. This innovative technology allows the investigation of protein activation as a consequence of protein-protein interaction or biochemical reactions, such as phosphorylation, glycosylation, ubiquitination, protein cleavage, and conformational alterations.Intracellular balance is carefully conserved by constant rearrangements of proteins through the activity of a series of kinases and phosphatases. Therefore, knowledge of the key cellular signaling cascades reveal information regarding the cellular processes driving a tumor's growth (such as cellular survival, proliferation, invasion, and cell death) and response to treatment.Alteration to cellular homeostasis, driven by elaborate intra- and extracellular interactions, has become one of the most studied fields in the era of personalized medicine and targeted therapy. RPMA technology is a valid tool that can be applied to protein analysis of several diseases for the potential to generate protein interaction and activation maps that lead to the identification of critical nodes for individualized or combinatorial target therapy.

Combined PDK1 and CHK1 inhibition is required to kill glioblastoma stem-like cells in vitro and in vivo

“…54 Briefly, cell lysates were diluted with 2 × Tris-Glycine SDS Sample Buffer (Life Technologies Corporation, Carlsbad, CA, USA) before printing on nitrocellulose slides (Grace Bio-Labs, Bend, OR, USA) and were spotted in triplicate with the Aushon 2470 contact pin arrayer (AushonBioSystems Inc., Billerica, MA, USA), in 4-point twofold dilution curves. All antibodies used in these experiments are listed in Supplementary Table S5.…”

Systems biology approaches to develop innovative strategies for lung cancer therapy

“…The latter involved the ‘pathway walking', in which a signaling cascade is mapped in body fluids using enzyme-linked immunosorbent assay, multiplex bead array assay or reverse-phase protein array of tumor samples or tissue microarray for immunohistochemical staining and the assessment of protein expression in tumors versus normal tissues. 8, 9 …”

Data Analysis Strategies for Protein Microarrays

“…This ensures the signal intensity is directly related with the specificity, the binding affinity and the sterical accessibility of the antibody against the target protein [7,9,14]. The production of a functional map for cell signaling pathways from individual cells or tissues by RPA arrays has increased the interest on this kind of arrays with the objective of developing personalized therapies [7,9,23]. The proteins involved in RPA do not require labeling and only a little amount of protein is needed to produce the microarrays.…”

“…Employing monoclonal antibodies in routine array production incurs excessive costs due to the hybridoma technology and their production. Therefore, phage display is an appropriate substitute according to the easier and cheaper methods to obtain and purify the antibody fragments (Table 2) [23,39,40]. …”