In recent years, mass spectrometric imaging (MSI) is frequently used to detect the biological molecules in the frozen tissue slice. However, it is difficult to detect the reactive metabolites such as GSH containing thiol group and small molecules with low mass. In addition, MSI needs the high-end instruments and takes a large amount of time to get molecular information. In this study, we developed a new system using thin-layer chromatography and Raman spectroscopy to detect GSH and Fe2+ in the frozen tissue slices easily.
First, we designed Raman probes for GSH and Fe2+ respectively. The Raman probe for GSH (MAL-EB) has maleimide as the reaction site for GSH and ethynylbenzene as the Raman tag. MAL-EB gives a highly polar GSH adduct upon the Michael reaction between maleimide moiety and thiol group of GSH. Therefore, after the separation of unreacted MAL-EB using low-polarity solvent, the Raman signals derived from ethynylbenzene (2110 cm-1) are observed in situ. On the other hand, the Raman probe for Fe2+ (Phen) forms an iron complex by chelating the Fe2+ ion. The complex shows absorption in the visible light region around 511 nm and Raman scattering signals (1455 cm-1) enhanced by resonance Raman effect. Thus, it is possible to detect the targets in the frozen tissue slice by monitoring each Raman signals simultaneously.
In fact, we applied this TLC tracing system to the mouse liver tissue. As a result, we found that it is possible to detect glutathione and Fe2+ in mouse frozen liver tissue with this system by monitoring the signals at 1455 cm-1 and 2110 cm-1.