Detection and characterization of selenoproteins by tandem mass spectrometry assisted by laser ablation inductively coupled plasma mass spectrometry: application to human plasma selenoproteins

Abstract

Accurate assessment of selenium status and selenium nutritional requirement in humans through the detection, identification and quantification of human plasma selenoprotein P (SelP) and glutathione peroxidase 3 (GPx3) has been an on-going effort and long term goal. Although several HPLC-ICP-MS analyses of human plasma/serum have reported SelP and GPx3 measurements, none of them have yet to demonstrate unambiguous mass spectrometry-based identification of these proteins. This study explored the potential of mass spectrometry techniques for the detection and identification of selenoproteins in a human plasma candidate Standard Reference Material (SRM) 1950 Metabolites in Human Plasma, with a total selenium concentration of 105.5 ± 2.3 ng g⁻¹. Since the classical proteomic shotgun approach of depleted human plasma is not specific and sensitive enough to identify low abundant selenoproteins, a laser ablation (LA) inductively coupled plasma mass spectroscopy (ICP-MS) method was developed. The challenge was to develop a highly sensitive method to target selenoproteins at physiological concentrations by using a conventional laser device and a sensitive LA-ICP-MS method easily transposable to other equipped laboratories. Through a combination of better sample preparation, by concentrating selenoproteins onto membranes, and increased sensitivity of Se detection, by humidifying ICP with an organic solution, a LA-ICP-MS method 80 times more sensitive compared to classical LA ICP-MS methods was developed. This method was successfully applied to the detection of SelP and GPx3 selenoproteins at physiological concentrations in samples of human plasma. Once detected, these low abundant selenoproteins were unambiguously identified by tandem mass spectrometry. This study highlights the importance of an approach combining ICP-MS and tandem mass spectrometry for unequivocal selenoprotein detection and identification at physiological concentrations. This procedure can be easily performed in other laboratories to study selenoproteins or other heteroatom-tagged proteins in humans or other biological samples using widely available instrumentation.