Dissecting protein function and signaling using protein microarrays

Abstract

Although many methods exist to study the recognition and signaling properties of proteins in isolation, it remains a challenge to perform these investigations on a system-wide or proteome-wide scale and within the context of biological complexity. Protein microarray technology provides a powerful tool to assess the selectivity of protein-protein interactions in high-throughput and to quantify the abundances and post-translational modification states of many different proteins in complex mixtures. Here, we provide an overview of the various applications of protein microarray technology and compare the strengths and technical challenges associated with each approach. Overall, we conclude that if this technology is to have a substantial impact on our understanding of cell biology and physiology, increased emphasis must be placed on obtaining rigorously controlled quantitative data from protein function microarrays and on assessing the selectivity of reagents used in conjunction with protein-detecting microarrays.

Figure 1

Common formats for protein microarrays. Protein function microarrays (a,b) are used to study molecular recognition and to identify protein–protein interactions, whereas protein-detecting microarrays (c,d) are used to detect and quantify proteins in biological samples. a, Microarrays of full-length proteins or protein domains are used to screen for protein–protein interactions (left); to study protein–peptide interactions (middle); or to identify antibodies in patient serum (right). b, Peptide microarrays are used to investigate protein–peptide interactions; to study substrate selectivity; and to profile enzyme activity in biological samples. c, Antibody microarrays are used to isolate proteins from complex mixtures. In the sandwich format (left), captured proteins are detected using a second, solution-phase antibody. In the direct-detection format (right), relative quantification is achieved by chemically labeling biological samples before applying them to the array. d, In contrast to antibody microarrays, lysate microarrays comprise the biological samples themselves. Complex mixtures of proteins are immobilized on nitrocellulose-coated glass substrates and detected using solution-phase antibodies. A labeled secondary antibody is often used to generate the signal.

Figure 2

Microarrays of protein domains for the quantitative study of molecular recognition. a, 159 SH2 and PTB domains were microarrayed in separate wells of a 96-well microtiter plate. The fluorescence arises from a trace amount of Cy5-labeled BSA that was added to each protein before arraying. b, SH2/PTB domain microarrays were probed with eight different concentrations of a fluorescently labeled phosphopeptide derived from ErbB2 (pTyr1139). c, Binding curves showing fluorescence as a function of peptide concentration for 28 high-affinity interactions. The data were fit to an equilibrium binding function to determine dissociation constants (K_D’s; blue text). This figure was adapted, with permission, from Jones et al. [10].