Comparative proteomics reveal distinct chaperone–client interactions in supporting bacterial acid resistance

Significance HdeA and HdeB are essential chaperones for many Gram-negative enteric bacteria to survive acid stress. How they cooperate to protect a broad range of client proteins from acid denaturation while avoiding nonspecific binding remains elusive. Here we developed a comparative proteomic strategy combining genetically encoded releasable photocross-linking with 2D difference gel electrophoresis, which allows an unbiased side-by-side comparison of the entire client pools from these two acid-activated chaperones in Escherichia coli . Our results reveal the existence of client specificities between these chaperones, and this pH-regulated client specificity mechanism demonstrates their seemingly paradoxical specific but promiscuous client recognition. Our comparative proteomic strategy provides a powerful platform for profiling protein–protein interactions under harsh conditions and for elucidating the otherwise elusive mode of action within native cellular contexts.