Use of laser-ablation inductively-coupled mass spectroscopy for analysis of selenosugars bound to proteins.

Abstract

We previously used high pressure liquid chromatography (HPLC) coupled with Se-specific inductively coupled plasma mass spectrometry (ICP-MS) and molecule specific (ESI Orbitrap MS/MS) detection to study the increase in liver Se in turkeys and rats supplemented as selenite in high-Se (5 µg Se/g diet) and adequate-Se diets. We found that far more Se is present as selenosugar (seleno-N-acetyl galactosamine) than is present as selenocysteine (Sec) in true selenoproteins. In high-Se liver, the increase in liver Se was due to low molecular weight (LMW) selenometabolites as glutathione-, cysteine- and methyl-conjugates of the selenosugar, but also as high molecular weight (HMW) species as selenosugars decorating general proteins via mixed-disulfide bonds. To demonstrate selenosugar binding to proteins, aqueous liver extracts from animals fed Se-adequate and high-Se were subjected to SDS-PAGE and Native-PAGE with and without pretreatment with β-mercaptoethanol (βME). The separated proteins were then electrophoretically transferred to membranes, and the membranes subsequently were subjected to laser-ablation inductively-coupled mass spectroscopy (LA-ICP-MS) analysis of 78Se profiles. Without βME treatment, 78Se was widely distributed across the molecular weight profile for both SDS-PAGE and Native-PAGE, whereas βME pretreatment dramatically reduced 78Se binding, reducing the profile to true Sec-selenoproteins. This reduction was ∼50% for both high-Se rat and turkey extracts. The increased 78Se in non-βME treated samples was distributed across the full profile. The use of LA-ICP-MS indicates that selenosugar residues are bound to protein subunits of multiple sizes, and that targeted attachment of selenosugars to a single or limited number of protein subunits does not occur.