Article Dans Une Revue Metallomics Année : 2025

Use of laser-ablation inductively-coupled plasma mass spectrometry for analysis of selenosugars bound to proteins

Résumé

Abstract We previously used high pressure liquid chromatography coupled with Se-specific inductively coupled plasma mass spectrometry 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 selenometabolites such as glutathione-, cysteine-, and methyl-conjugates of the selenosugar, but also as high molecular weight species as selenosugars decorating general proteins via mixed Se-S bonds. To demonstrate selenosugar binding to proteins, aqueous liver extracts from animals fed Se-adequate and high-Se were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (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 plasma mass spectrometry (LA-ICP-MS) analysis of 78Se profiles. Without βME treatment, Se was widely distributed across the molecular weight profile for both SDS-PAGE and Native-PAGE, whereas βME pretreatment dramatically reduced Se binding, reducing the profile to true Sec-selenoproteins. This reduction was ∼50% for both high-Se rat and turkey extracts. The increased Se 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.

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Dates et versions

hal-04968914 , version 1 (27-02-2025)

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Katarzyna Bierla, Joanna Szpunar, Ryszard Lobinski, Roger Sunde. Use of laser-ablation inductively-coupled plasma mass spectrometry for analysis of selenosugars bound to proteins. Metallomics, 2025, 17 (2), ⟨10.1093/mtomcs/mfaf002⟩. ⟨hal-04968914⟩
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