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WIREs Syst Biol Med
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Protein microarrays for genome‐wide posttranslational modification analysis

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Abstract Protein microarray technology has emerged as a powerful tool for comparing binding interactions, expression level, substrate specificities, and posttranslational modifications (PTMs) of different proteins in a parallel and high‐throughput manner. The ability to immobilize proteins to a solid surface and register the specific address of each protein has bridged major limitations for investigating the proteome in biological samples, namely, the wide dynamic range of protein concentrations and the perturbation of the physical and chemical properties of proteins by their modification. Recent advances introduced the use of functional mammalian cell extracts to assay PTMs under different cellular conditions. This assay offers a new approach for performing large‐scale complex biochemical analysis of protein modifications. Here, we review studies of PTM profiling using protein microarrays and discuss the limitations and potential applications of the system. We believe that the information generated from such proteomic studies may be of significant value in our elucidation of the molecular mechanisms that govern human physiology. WIREs Syst Biol Med 2011 3 347–356 DOI: 10.1002/wsbm.120 This article is categorized under: Laboratory Methods and Technologies > Proteomics Methods

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A portion of a microarray. Several different duplicate proteins and their color‐coded corresponding signal intensity values are presented. The microarray was labeled with an α‐polyubiquitin antibody and a fluorescently labeled secondary antibody was used for detection. Spots that are black reflect a nonreactive/unmodified protein.

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Potential applications for extract‐ and tissue‐based functional assays. Different sample types (i.e., patient tissue or cell culture extracts) may be used to identify targets of different posttranslational modifications (PTMs) (e.g., acetylation, phosphorylation, ubiquitination, etc.) on a genome‐wide scale. PTM profiling may be used to study different cellular processes as well as physiological processes that may be important for our understanding of human disease.

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Visual inspection of differential ubiquitination. Microarrays were incubated with mitotic extracts with or without the addition of the C‐terminus of emi1 (anaphase promoting complex inhibitor) for 60 minutes at room temperature. Each duplicate spot represents one protein and a few different examples were chosen (e.g., Tyro3, Aurora kinase C, Nek2) to show the different reactivity levels that were registered for these proteins under the two experimental conditions. The signal intensity varies from low reactivity (black spot) to high reactivity (green).

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Testing the reproducibility of the assay. Microarrays were incubated with mitotic extracts supplemented with UbcH10 for 60 minutes at room temperature. The polyubiquitination signal per protein was calculated by the mean values of its spots reactivities minus its local background intensity. Scatter plots of the signal intensities on each microarray were plotted to compare the variability between duplicate spots on the same array (left) and of technical replicates of two different microarrays developed under the same conditions (right). The correlation coefficient of the two comparisons is given in the inset.

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Identifying meaningful reactivities based on the signal‐to‐noise ratio (SNR). The SNR value of each spot on the array was calculated by the signal intensity minus the local background divided by the standard deviation of the background. The signal intensity of each spot on the array (y‐axis) is plotted as a function of its SNR value (x‐axis). An SNR of > 3 (red line) is considered as a positive signal (i.e., above background reactivity).

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