A Quantitative Proteomics Approach to the Identification of Protein Targets of Small Bacterial RNAs

Residue-specific incorporation of non-canonical amino acids (ncAAs) into cellular proteins allows discrimination of new proteins from old; labeled proteins can be selectively conjugated to purification tags, enriched by affinity chromatography, and identified by mass spectrometry. This method, termed bio-orthogonal non-canonical amino acid tagging (BONCAT), is an attractive approach for the selective identification of proteins produced by a cell in response to biological cues. In this work we combined the BONCAT approach with stable isotope labeling by amino acids in cell culture (SILAC), to develop a proteomic technique capable not only of enriching but also of quantifying the subset of newly synthesized proteins produced during an ncAA pulse. Armed with this method, we conducted a proteomic search for direct regulatory targets of the E. coli small RNA CyaR, a Crp-activated regulatory RNA involved in cell metabolism and quorum sensing. Following expression of either CyaR or a negative RNA control, we pulse-labeled cultures with azidohomoalanine (Aha), a methionine surrogate, for 15 minutes. Aha-labeled proteins were conjugated to an alkynyl-biotin affinity tag by copper-catalyzed azide-alkyne cycloaddition, purified on streptavidin resin, and analyzed by LC/MS-MS. By comparing the relative abundances of proteins produced in the CyaR and control cultures, we identified three known CyaR protein targets and a set of 26 candidate proteins as significantly up or down-regulated. A regulatory RNA assay with target-GFP fusions verified a subset of the candidate proteins as directly regulated by CyaR.