Abstract
Microarray-based proteomics expanded the information potential of DNA arrays to the level of protein translation and interaction, but so far, not much beyond. Although enzymatic activity from immobilized proteins has been reliably studied using surface plasmon resonance, a microarray of catalytically competent enzymes would facilitate high throughput, parallel study of their function. The ability to localize activity from soluble substrates has frustrated development of such an array. Here, we report the novel use of previously developed, highly specific suicide substrates for three families of enzymes: deubiquitylases, deSUMOylases, and deISGylases. We show specificity of each family to its cognate substrate, and demonstrate utility of the array in a secondary screen of small molecule inhibitors.
Highlights
Microarray-based proteomics has typically sought to accomplish the same primary objectives as its accomplice, mass spectrometry (MS): cataloging protein expression, defining protein-protein interactions, and defining types and sites of post-translational modification [1]
We found high degrees of specificity for various substrates based on published activities (e.g. DUBs cleave UB, sentrin-specific proteases (SENPs) cleave SUMO, deISGylases cleave ISG), and report a novel deISGylase activity from a previously identified DUB
Activity of DUBs was measured by detecting ubiquitin vinyl methylester (UBVME) on the array by immunoblot with anti-ubiquitin
Summary
Protein Production—DNA clones were obtained from OpenBio Systems (Huntsville, AL). Protein coding regions were PCR amplified and subcloned into bacterial expression vectors. Following shaking the third PBST wash from the arrays, rabbit anti-ubiquitin (Sigma U5379) diluted 1:9 in PBST was applied to each subarray and incubated 1 h at RT. Following washing three times with PBST as above, arrays were incubated with FITC conjugated goat anti-rabbit (Jackson ImmunoResearch 111-095-003) 1:49 in PBST for 1 h at RT. The normalized data from six printed spots of each DUB/dilution combination were summarized by their median, and standard deviation calculated. Nonarray UBVME Labeling Experiments—Solution-based labeling was done under conditions identical to the array based experiments, except that isopeptidases were diluted to 2 M in PBS plus inhibitor (or mock inhibitor). P1001 is compound NSC119889 from the Open Chemical Repository, Developmental Therapeutics Program (NCI/NIH: http://dtp. cancer.gov), and was diluted and applied to microarrays directly by Progenra employees who maintained control of the sample at all times
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