Simultaneous electrothermal vaporization and nebulization sources and improved methodologies for metallomic studies using ICP-MS
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Both electrothermal vaporization (ETV) and nebulizer introduction sources offer unique advantages for inductively coupled plasma mass spectrometry (ICP-MS) analyses. A device for coupling the ETV and nebulizer was developed so that a quick switch from the nebulizer to the ETV (termed 'inline-ETV') could help gain additional information. The inline-ETV produced similar limits of detection (LODs) for most elements in both HNO₃ and HCl matrices compared to a conventional nebulizer or ETV. However, in a problematic matrix, isobaric interferences could exist that may not be accounted for in a typical nebulizer analysis. In a 1% HCl matrix, the LODs for ⁵¹V and ⁵³Cr--which are interfered with by ⁵¹ClO⁺ and ⁵³ClO⁺, respectively--improved 65- and 22-fold using the inline-ETV source compared to a typical nebulizer. In recent applications, ICP-MS has gained attention as a way of determining metal-protein associations. A novel broad-based methodology was developed to characterize metal-protein associations. The method utilized native gel electrophoresis for separation followed by electroblotting onto chemically-modified quartz membranes. The membranes were analyzed for metals using laser ablation ICP-MS. Modified membranes were shown to improve sensitivity compared to ablating a dried gel directly or using a commercially-available membrane. The coupling of separation by preparative ultracentrifugation and metal detection by ICP-MS was explored for metal-protein equilibrium determinations. This study characterizes the stoichiometry as well as apparent (K[subscript app]) and intrinsic (K[subscript int]) binding affinities for Cu-BSA, which was used as a model protein. K[subscript app] and K[subscript int] were determined at two different conditions, pH 9.53 and pH 7.93 in 100mM Tris buffer. The pH-independent K[subscript int] value at pH 9.53 agreed closely with literature values, while the value at pH 7.93 was approximately 2.5x larger. BSA undergoes a structural rearrangement between pH 7-9, and the generally accepted pH-dependency of protein tertiary structure may be responsible for the variations in the "intrinsic" binding constant. Overall, this study validates and shows the efficacy of combining preparative ultracentrifugation with ICP-MS detection for interrogating metal-protein associations while causing minimal equilibrium perturbations as a result of the separation and measurement processes.