Biarsenical-based fluorescent labeling of metallothioneins as a method for ultrasensitive quantification of poly-Cys targets

Abstract

Background

Mammalian metallothioneins (MTs) play a crucial role in maintaining Zn(II) and Cu(I) homeostasis, as well as regulating the cellular redox potential. They are involved in cancer resistance to cisplatin-related drugs and the sequestration of toxic metal ions. To investigate their participation in specific physiological and pathological processes, it is imperative to develop an analytical method for measuring changes in protein concentration both in vitro and in vivo. Over the years, numerous methods have been developed for this purpose; however, they often suffer from issues such as low sensitivity, lengthy sample preparation, or variability in antibody batches.

Results

In this study, we present, for the first time, the application of the fluorescent biarsenical probe CrAsH-EDT₂ for the specific and ultrasensitive detection of human MTs. The interaction between the small library of biarsenical probes and 11 human MT isoforms (MT1-MT4), isolated MT2 domains, snail LlMT, and plant phytochelatin PC4 was investigated using spectrofluorimetry and mass spectrometry. A key advantage of our method is that the probe excess does not need to be removed before measurement as unbound is virtually non-fluorescent. We established protocols for MT detection in various assays, including cuvette-based, SDS-PAGE, and capillary electrophoresis. A detection limit (LOD) of 100 fg for human MT2 was achieved. Finally, we observed increased metallothionein concentration in lysates of HeLa cells treated with ZnSO₄.

Significance

In conclusion, our results demonstrate that the established method is easy to apply, versatile, and can be adapted to detect metallothioneins and other poly-Cys targets in different assays and applications.