Detection of metals in proteins by means of polyacrylamide gel electrophoresis and laser ablation‐inductively coupled plasma‐mass spectrometry: Application to selenium

Abstract

The capabilities of laser ablation-inductively coupled plasma-mass spectrometry for the detection of trace elements in a gel after gel electrophoresis were systematically studied. Figures of merit, such as limit of detection, linearity, and repeatability, were evaluated for various elements (Li, V, Cr, Mn, Ni, Cu, Zn, As, Se, Mo, Pd, Ag, Cd, Pt, Tl, Pb). Two ablation strategies were followed: single hole drilling, relevant for ablation of spots after two-dimensional (2-D) separations, and ablation with translation, i.e., on a line, relevant for one-dimensional (1-D) separations. This technique was applied to the detection of selenoproteins in red blood cells extracts after a 1-D separation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and the detection of selenium-containing proteins in yeast after 2-D electrophoresis (2-DE). The detection procedure was further improved by using the dynamic reaction cell technology, which allowed the removal of the Ar 2 interference and hence the use of the most abundant Se isotope, 80 Se. Reaction gases were compared (methane, carbon monoxide, ammonia, oxygen and the combination of argon (collision gas) and hydrogen (reaction gas)). In each instance, the reaction cell parameters were optimized in order to obtain the lowest detection limit for Se (as 80 Se 16 O 1 with O 2 as the reaction gas). Carbon monoxide was found to offer the best performance. The detection limit with the use of DRC and He as transport gas was 0.07 mg Se g 21 gel with single hole drilling and 0.15 mg Se g 21 gel for ablation with translation.

Evaluation of gel electrophoresis conditions for the separation of metal‐tagged proteins with subsequent laser ablation ICP‐MS detection

“…The reproducibility of scans over the whole concentration range of the element-doped gels was between 5 and 20% (five repeats of 2 mm long scans) and correlation coefficients for calibrations using metal-doped gels on each occasion were better than 0.99 between 0.2 and 2.0 mg element/kg wet gel. LODs reported by Chery et al using a 193 nm LA system are in a concentration range similar to ours [32], although Zn detection appears to be slightly more sensitive with our system, while the LOD of Cu are slightly lower using the 193 nm laser system and He as the transport gas. The LODs could, however, be improved if an ICP-SF-MS [21] were to be used or a femtosecond laser (with which more material can be ablated compared with nanosecond lasers in the same time), as recently shown [33].…”

The role of cisplatin and NAMI-A plasma-protein interactions in relation to combination therapy

“…ICP-MS is a powerful and highly sensitive technique for elemental analysis, and has been widely used when interfaced with HPLC for the analysis of metals in biological samples (30,31). Combined with laser ablation, ICP-MS has been used to analyse metallo-, seleno-or phospho-proteins resolved by PAGE (26,27,32,33).…”

Apport de la Technique ICP-MS à l'analyse toxicologique : la spéciation des métaux

“…Enfin, une application particulière est fondée sur l'utilisation de l'ablation laser et son usage pour l'identification de nouvelles protéines comportant un ou plusieurs atomes métalloïdiques tels que le sélénium par exemple. Cette étude est réalisée par l'ablation sur un gel électrophorétique, éventuellement bidimensionnel (15,16), après séparation des différentes protéines en fonction de leur poids moléculaire. La figure 10 montre ainsi l'intérêt de l'ablation laser dans la démonstration de la présence de Se dans une protéine spécifique préalablement séparée par ICP-MS (41).…”