Species specific isotope dilution versus internal standardization strategies for the determination of Cu, Zn-superoxide dismutase in red blood cells

Abstract

Many proteins (more than one third) contain metal ions either within their own structures or bound to some of their active sites. These metals are involved in numerous biological processes and therefore, the quantification of metalloproteins that can serve as clinical biomarkers is of great interest. With this aim, the development of analytical strategies that permit individual (targeted) protein quantitative analysis is attempted in this work. In particular, the evaluation of different strategies for the determination of Cu, Zn-superoxide dismutase (Cu, Zn-SOD), a metalloprotein present in the first-line antioxidant defence system of the body, is conducted. The first analytical strategy is based on the use of bovine Cu, Zn-SOD as internal standard for the quantitative analysis of human Cu, Zn-SOD. For this aim, the chromatographic separation between both species (bovine and human) has been optimized according to their respective isoelectric point by anion exchange chromatography. Interestingly, the obtained results revealed a faster specific degradation of the bovine standard with respect to human SOD during sample preparation. The second strategy involves the production and evaluation of an isotopically enriched metalloprotein standard to be used as tracer in the species specific isotope dilution (SS-IDA) method by measuring the Cu associated to the protein. This is done by liquid chromatography with online inductively coupled plasma mass spectrometric (ICP-MS) detection and applied to the quantification in bovine erythrocytes. This finding is a good example to illustrate the power of SS-IDA for targeted protein quantification in respect to the commonly used alternative standards.

Quantification of ferritin bound iron in human serum using species-specific isotope dilution mass spectrometry

“…So far, only a very limited number of attempts have been made to employ SS-IDMS for quantification of metallo-proteins. 24,[37][38][39][40][41][42][43][44] The developed technique for measuring FBI in serum may serve as an example to further illustrate the potential of SS-IDMS in biomedical research and the possibilities arising from its use to identify and study new biomarkers in health and disease.…”

Update on the status of metrology for metalloproteins

“…In the case of SOD1 the metals can be chemically removed 41,42 and replaced with their isotopically enriched form. 43,44 This method is a good starting point for the development and validation as a reference measurement procedure for SOD1 in human erythrocyte lysate which was successfully conducted within the EMRP project HLT05 by Gleitzmann et al 45 As discussed before this approach is less prone to matrix effects and interferences compared to external calibration or activity assays and is, therefore, more promising than other approaches.…”

Fluorescent Biosensors Based on Silicon Nanowires

“…This interest can be ascribed to the possibilities offered by nanotechnology to improve nanomaterial physical properties in combination to the high surface-to-volume ratio of nanostructures, permitting them to overcome the standard sensor limits [38][39][40][41][42]. Among all the nanomaterials, thanks to their robust photostability, efficient fluorescence at tunable wavelength, semiconductor light-emitting quantum dots (QDs) are typically known as highly fluorescent nanoprobes and have been largely applied in imaging, as well as in biosensing applications [43][44][45][46][47][48][49]. Nonetheless, QDs often are characterized by a complex synthesis that results incompatible with industrial processes.…”