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Identification of metal-associated proteins in cells by using continuous-flow gel electrophoresis and inductively coupled plasma mass spectrometry.

Metals and metalloids are crucial for life and indispensable for a series of biological processes. It is estimated that a quarter to one third of all proteins require metals to carry out their functions, and roughly half of the known enzymes uses a particular metal as a cofactor. In spite of the prevalence and importance of metalloproteins, they are generally poorly characterized in many organisms. A recent study demonstrated that the microbial metalloproteome is much more extensive and diverse than we presently know. Currently, it is impossible to predict, genome-wide, the numbers and types of metals used by organisms and to define any metalloproteome until the proteins are fully characterized owing to diverse and poorly recognized metal coordination sites. Moreover, metals/metalloids have long been used for therapeutic purposes, for example, arsenic trioxide for the treatment of acute promyelocytic leukemia. The detailed molecular mechanisms, however, are still not fully understood owing to the complex functions of metals in biological systems. A robust and convenient approach, by which metals/metalloids can be mapped to their associated proteins proteome-wide is urgently needed. Such a methodology will improve our understanding of the molecular mechanisms of metal-dependent biological processes and profoundly promote metallomics research, an integrated biometal science complementary to genomics and proteomics. Gel electrophoresis has been one of the commonly used methods for separation and analysis of proteins based on their molecular mass and charge; however, it fails to provide information on metal identity and content for metalloproteins. The lack of convenient subsequent methods for specific metal detection confines its application on providing metalrelated information of corresponding proteins. Although laser ablation inductively coupled plasma mass spectrometry (LAICP-MS) and synchrotron X-ray fluorescence spectrometry (SXFS) have been used for the identification of metalbinding proteins on gels and in tissues/organs, either compromised sensitivity originating from the sample introduction system or limited accessibility to the synchrotron facility prevents their routine applications. Other strategies such as metal isotope radioautography, which employs unique metal isotopes to label metalloproteins, are also very inconvenient for daily usage. Herein, a new strategy based on column-type gel electrophoresis coupled with a metal-specific detection system, that is ICP-MS, was developed (Figure 1a), allowing both metals and their associated proteins to be examined comprehensively. Since the strategy can be used to analyze and at the same time to separate and isolate proteins, it can readily be applied to not only detect metalloproteins and/or metalbound proteins with a sensitivity at the femtomole level, but also conveniently integrate current proteomics with metallomics. We further showed the bismuth profile in cell lysates of Helicobacter pylori upon treatment with colloidal bismuth subcitrate (CBS) and further characterized metal-binding features of H. pylori SlyD (HpSlyD) inside cells. The column-type gel adopted the traditional slab gel preparation. Both native and denaturing conditions could be applied, and the gel compositions varied with the protein targets of interest. To validate the feasibility of the column gel system, three metal-binding proteins, Cu-bound bovine serum albumin (Cu-BSA), Cu,Zn superoxide dismutase (Cu,ZnSOD), and diferric transferrin (Fe2-Tf), were mixed and subjected to separation. Three bands, corresponding to Fe2Tf, Cu,Zn-SOD, and BSA, were visualized on a CoomassieBlue-stained slab gel (Figure 1b). The proteins separated by column-type gel electrophoresis gave rise to migration profiles similar to those observed in classical slab gel under comparable conditions. The elutes from the column gel system were split into two parts by using a T connection, with one for online metal measurement by ICP-MS and the other for protein identification through biological mass spectrometry analysis of the collected fractions (Figure 1a). It is noted that one peak was observed in either the Zn or Fe profile corresponding to SOD and transferrin, respectively, indicative of association of Zn ions with SOD and binding of Fe ions to Tf; whereas there are two peaks in the Cu profile, with each corresponding to a distinct molecular mass, thus suggesting that copper binds to both SOD and BSA. The amounts of metals were quantifiable (Figure S2 in [*] Dr. L. Hu, Dr. T. Cheng, Dr. B. He, Y. Wang, Y.-T. Lai, Prof. H. Sun Department of Chemistry, The University of Hong Kong Pokfulam, Hong Kong (P. R. China) E-mail: hsun@hku.hk