Investigation of the Low Molecular Weight Thiol Composition in a Metastatic Prostate Cancer Cell Line (LNCaP) by LC-UV-MS and NMR after Labelling with the Ellman Reagent

Abstract

The low molecular weight thiols present in the deproteinized extract of a prostate cancer cell line (LNCaP-FGC) were analysed after derivatization with the Ellman reagent (ESSE). The mixed disulphides formed (RSSE) were fractionated, characterized and quantified by liquid chromatography on a C-18 column using UV detection. This revealed the presence, in femtomoles per cell, of glutathione (8.30 ± 0.73), cysteine (2.71 ± 0.04) and cysteinylglycine (0.83 ± 0.10), accounting for the bulk of the thiol present. Further analysis of the cell extracts using a novel and sensitive mass spectrometry technique allowed the detection of low level of an additional derivative which was identified as cysteinylglycerate using NMRspectroscopy.

Isolation and analysis of a non-protein low molecular weight thiol-mercurial adduct from human prostate lymph node cells (LNCaP)

“…The thiol contents determined using the Ellman reagent on the cellular fractions obtained in the present (as shown in Figure 2) and previous studies [6,7] are given in Table 1 below. In earlier studies, the G15 protein thiol concentration was calculated from the adduct obtained from the Ellman reagent.…”

“…Recent work on a human prostate cancer cells, a lymph node line known as LNCaP, has shown that thiols other than GSH are present in the deproteinized extract (acid soluble fraction -ASF) of these cells [5,6]. Following up this work, investigations have focused on the protein thiol content of the chromatin cellular pellet left after removal of the ASF which was found to contain 76.7% of the cellular thiol (43.2 + − 2.9 fmol/cell).…”