Elemental mass spectrometry for Se-dependent glutathione peroxidase determination in red blood cells as oxidative stress biomarker

Abstract

Glutathione Peroxidase-1 (GPx1) is an enzyme playing an important role in the defense against oxidative stress which is associated with many pathological conditions. Thus, changes in the expression of this enzyme in different human tissues and fluids could be an indicator used for oxidative status assessment. Since most analytical methods for GPx1 determination are based on relative activity measurements, here we propose the absolute quantification of this protein in red blood cells through the measurement of Se present as selenocysteine in its structure. For this purpose, the sample is treated for hemoglobin precipitation and further fractionated by size-exclusion liquid chromatography (SEC) with ICP-MS for final detection. Purity of the species in the fraction obtained by SEC was assessed by orthogonal chromatographic separation based on a reversed phase mechanism (RP) of the peptides obtained by tryptic digestion of the corresponding fraction. The use of ICP-MS detection after such capillary-RP-HPLC permitted us to detect a single Se-containing peptide that was further confirmed by electrospray ionization mass spectrometric detection (ESI-MS) to belong to GPx1. Once the purity of such fraction was addressed, the quantitative analysis of GPx1 was conducted by Se post-column isotope dilution analysis after SEC separation in different samples of human red blood cells. When the concentration results obtained via SEC-ICP-MS for different protein standards are plotted versus the activity measurements (using the spectrophotometric H₂O₂/NADPH/GR method) a good correlation curve is obtained. Such results permit us, from ICP-MS measurements, to obtain simultaneously the GPx1 absolute concentration as well as the activity by interpolation in the previously obtained curve.