Identification and quantification of seleno-proteins by 2-DE-SR-XRF in selenium-enriched yeasts

Abstract

A comprehensive approach that can identify and quantify selenium (Se) in seleno-proteins in Se-enriched yeast was developed. The Se-containing compounds in Se-enriched yeast were first extracted and then the fraction of Se-containing proteins in the supernatant was analyzed by 2-dimensional electrophoresis (2-DE) and synchrotron radiation X-ray fluorescence (SR-XRF). The detection limit (DL) of SR-XRF analysis for Se quantification in Se-containing proteins after 2-DE separation was calculated to be 0.20 µg g⁻¹, which is suitable for Se quantification in the Se-containing spots present on the 2-D gel. After being scanned by SR-XRF, only spots with a mean Se content exceeding twice the DL of SR-XRF were considered to be seleno-proteins. In this way, a total of 157 Se-containing spots in the gel were visually distinguished. Se contents in all the Se-containing proteins of different molecular weights were quantified. The total Se content on the 2-D gel was calculated to be 126.56 µg g⁻¹, which covered most of the seleno-proteins on the 2-D gel.