Integration of fluorescence imaging with proteomics enables visualization and identification of metallo-proteomes in living cells

Metalloproteins account for nearly one-third of proteins in proteomes. To date, the identification of metalloproteins relies mainly on protein purification and the subsequent characterization of bound metals, which often leads to losses of metal ions bound weakly and transiently. Herein, we developed a strategy to visualize and subsequently identify endogenous metalloproteins and metal-binding proteins in living cells via integration of fluorescence imaging with proteomics. We synthesized a "metal-tunable" fluorescent probe (denoted as Mⁿ⁺-TRACER) that rapidly enters cells to target proteins with 4-40 fold fluorescence enhancements. By using Ni²⁺-TRACER as an example, we demonstrate the feasibility of tracking Ni²⁺-binding proteins in vitro, while cellular small molecules exhibit negligible interference on the labeling. We identified 44 Ni²⁺-binding proteins from microbes using Helicobacter pylori as a showcase. We further applied Cu²⁺-TRACER to mammalian cells and found 54 Cu²⁺-binding proteins. The strategy we report here provides a great opportunity to track various endogenous metallo-proteomes and to mine potential targets of metallodrugs.