Reverse phase protein arrays

Antigen arrays, also described as reverse-phase protein arrays (Paweletz et al., 2001), involve the immobilization of proteins to serve as bait for various protein-protein interactions (Sreekumar and Chinnaiyan, 2002). For example, Joos et al. (2000) printed down various autoantigens present in sera with known associations with various autoimmune diseases such as Graves disease lupus connective tissue disease, and others. The group then screened various sera for the presence of autoantibodies. By immobilizing on the array a serial dilution series for each antigen, the titers for these antibodies could be determined. 

The term reverse phase protein microarray was first coined in 2001 by Paweletz et al. (5) in the landmark paper which introduced the platform and demonstrated its utility for analyzing proteins derived from laser capture microdissected (LCM) prostate cancer. RPMAs have also been referred to by other names in the literature including lysate arrays, reverse phase lysate arrays, protein microarrays, and reverse phase protein arrays (6). A very nice historical perspective on the development of RPJMA technology is presented in a review by Mueller et al. (6). A detailed description of the methods for performing RPMA is beyond the scope of this chapter and would be redundant because there are... 

Wilson, B. Monitoring proteins and protein networks using reverse phase protein arrays... 

Brase JC, Mannsperger H, Sultmann H et al (2011) Antibody-mediated signal amplification for reverse phase protein array-based protein quantification. Methods Mol Biol 785 55-64... 

Brase JC, Mannsperger H, Frohlich H et al (2010) Increasing the sensitivity of reverse phase protein arrays by antibody-mediated signal amplification. Proteome Sci 8 36... 

Martiny-Baron G, Haasen D, D Dorazio D et al (2011) Characterization of kinase inhibitors using reverse phase protein arrays. Methods Mol Biol 785 79-107... 

Hennessy BT, Lu Y, Gonzalez-Angulo AM et al (2010) A technical assessment of the utility of reverse phase protein arrays for the study of the functional proteome in non-microdis-sected hiunan breast cancers. Clin Proteomics 6 129-151... 

Reverse-Phase Protein Arrays-Based Studies. 113... 

Reverse-phase protein arrays offer a robust new method of quantitatively assessing expression levels and the activation status of a panel of proteins. For this purpose, the lysate of protein(s) of interest is arrayed without selection via a capture molecule. This array can then be queried with an antibody or ligand probe, or an unknown biological component. Since an individual test sample is immobilized in each array spot, this array can be composed of a variety of different patient samples. Each array is incubated with one detection protein or antibody, and a single end point is measured across the arrayed cohort and can be directly compared across multiple samples. Replicates can be reproducibly printed at a given sitting, increasing quality control over a series of queried arrays (reviewed in [33]). 

Grubb RL, Calvert VS, Wulkuhle JD, Paweletz CP, Linehan WM, Phillips JL, et al. Signal pathway profiling of prostate cancer using reverse phase protein arrays. Proteomics 2003 3(11) 2142—2146. 

Utility of reverse phase protein arrays applications to signalling pathways and human body arrays. Brief Fund. Genomic Proteomic 1, 305-315. 

In contrast to sandwich immunoassays, in which a capture antibody is immobilized on a substrate ( forward phase arrays ), in the so-called reverse phase protein arrays the complex mixture of analytes is immobilized on a substrate. The biological sample could be a cell lysate or from another source. Each spot then comprises the entire proteome of a biological system at a certain state or after a certain treatment. Each array contains a multitude of biological states. Several copies of such arrays are produced and each one is probed with a single antibody. Thus, each assay shows, in one experiment, the expression levels of a particular protein in all the different biological states represented on the array. Since the entire proteome is contained in each spot, essentially any protein can be analyzed in the context of different experimental foci. 

FIGURE 1 Major types of protein microarrays (A) targeted protein arrays, (B) reverse-phase protein arrays, and (C) in situ expressed protein arrays. 

Proteomics, protein abundance, function, and interactions within the cell, can be measured by liquid chromatography (6) and mass spectrometry or by reverse-phase protein arrays. The interactions are done by yeast-two-hybrid, cochromatography, and other methods. 

Wilson, B., L. A. Liotta, and E. Petricoin, 3rd. 2010. Monitoring proteins and protein networks using reverse phase protein arrays. Dis. Markers 28 225-32. 

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