Tracking Endogenous Metalloproteins by Metal Chelation Based Fluorescence Approach

Abstract

Metalloproteins, which account nearly a quarter to one third of human proteomes, are involved in various biological functions. In particular, metalloproteins play critical roles in metal homeostasis and disease processes. Moreover, they often serve as drug targets especially for metallodrugs.1 Despite of numbers of techniques e.g. ICP-MS, or synchrotron-based techniques that have been employed for identification of metalloproteins,2 conventional method, which involved in the protein separation or purification followed by detection of metals, often results in the loss of metal ions that bind to a protein weakly or transiently. We report the design and synthesis of a new family of metal-chelation based fluorescence probes, consisting of a metal-binding moiety, a fluorophore and arylazide.3-6 The metal-binding moiety will guide the probe to bind to proteins in live cells, and arylazide serves as an anchor, upon photoactivation, covalent bonds between the probe and labelled proteins will be formed, enabling downstream protein identification through conventional proteomics. We have developed a series of metal tunable NTA-based fluorescence probes, and identified various metallo-proteomes including Bi(III), Fe(III), Cu(II) and Ni(II) proteomes in various prokaryotic and eukaryotic cells, which provide a basis for further analysis of the biological pathways that these metals participate as well as the mode of action of metallodrugs. Moreover, by conjugation of arsenic binding moiety with a fluorophore and arylazide, an organoarsenic probe was also developed. Using this probe, a number of arsenic-binding proteins have been tracked in both NB4 and HL60 cells. In combination with quantitative proteomics, a new protein target of arsenic trioxide was identified and validated, which extend our knowledge on the mechanism of action of arsenic trioxide. The methodology we developed here combined with other (metallo)proteomics will further advance our understanding on the roles of metals in biology and medicine.