Quantification and visualization of glutathione S-transferase omega 1 in cells using inductively coupled plasma mass spectrometry (ICP-MS) and fluorescence microscopy

摘要

We report a novel activity-based and Cu-free click chemistry (CC) mediated methodology for glutathione S-transferase omega 1 (GSTO1) quantification using species-unspecific isotope dilution inductively coupled plasma mass spectrometry (SUID ICP-MS), in which dibenzylcyclooctyne-modified 2-chloroacetamide (DBCO-ChAcA) was designed and synthesized, meanwhile, as a navigator towards GSTO1 for subsequent N-3-DOTA-Eu-tagging via Cu-free CC. Using Eu-153-SUID ICP-MS coupled with size exclusion chromatography (SEC), the LOD (3 sigma) of GSTO1 reached 6.9 fmol with an RSD of 2.4 % at the 0.1 mu M level (n=5) considering the recovery of GSTO1 on the SEC was 96.5 +/- 2.4 %. The GSTO1 contents in the cells of human hepatocellular carcinoma C7721 and breast carcinoma MCF-7 as well as normal hepatic C7701 without or with cis-platin administration were quantified to be from 1.2 mu g/10,000 cells (n=3, RSD=4.5 %) corresponding to 1.2x10(-2) ng per cell to 4.76 mu g/10,000 cells (n=3, RSD=2.9 %) corresponding to 4.76x10(-2) ng per cell. For a comparative study, DBCO-ChAcA-fluor 488-based fluorescence microscopy could not alone visualize GSTO1 in the cells but could together with those from the small SH-containing molecules such as GSH and that from extra N-3-fluor 488 in the cells. This activity-based CC-mediated tagging/labeling strategy provided an opportunity for ICP-MS-based targeted protein quantification, and is very much expected to find its applications in biological mechanism study and the subsequent drug design.