Reference measurement procedures for the iron saturation in human transferrin based on IDMS and Raman scattering

Abstract

Two reference measurement procedures are presented here that allow the determination of the iron saturation in human transferrin, based on different molecular properties. The results, directly derived from the number of ions bound to the protein molecule, are traceable to the SI. Up to now, the iron saturation has only been deduced indirectly from the amount-of-substance ratio of serum iron to transferrin in serum. Interlaboratory tests have shown the need for more accurate methods, as the results from many participant test samples for both parameters do not lie within the acceptable range of deviation given by relevant guidelines when different methods or kits are applied. Using isotope dilution, an HPLC ICP-MS procedure was developed in compliance with the requirements of a primary reference measurement procedure. In this manner, the iron saturation was measured with an associated relative expanded measurement uncertainty of 4%. Based on the results, a straightforward Raman procedure was evolved, which allows the determination of the iron saturation in transferrin with an associated relative expanded uncertainty of 7%.

Step‐by‐step monitoring of a magnetic and SERS‐active immunosensor assembly for purification and detection of tau protein

“…The simplified preparation concept for a controlled formation for a SERS‐active immunosensor greatly improves the applicability of this approach and allows a larger scale implementation. Furthermore, a quantification principle will be established utilizing the isotope dilution (ID) technique, which is mostly used in mass spectrometry, but which has also been shown to work excellently when combined with Raman spectroscopy . Besides the advantages of having molecular specificity, accuracy and a high sensitivity during quantification, an ID‐SERS approach also could provide SI‐traceability of the measurement results when combined with well‐characterized reference materials.…”

Update on the status of metrology for metalloproteins

“…A species specic spike material was produced and thoroughly characterised regarding its protein structure, Fe load and isotopic composition. 16,17 A similar approach was developed by Ordonez et al, who besides total TRF also quantied the sialoforms using postcolumn ID-ICP-MS. 18 Both methods shall be used for the certication of the SRM-909c reference material in future. To reduce the inuence of Fe background and low isotopic enrichment in the spike material, triple isotope dilution mass spectrometry (IDMS) was used for the quantication of total TRF.…”

Metrological Approach for the Quantification of Metalloproteins (EMRP Project HLT05)

“…As only the metal is detected in ICP-MS, a complete saturation of the protein has to be ensured for the successful application of IDMS for the quantification of the metalloproteins. A cost and time saving method to determine the iron saturation in TRF was developed by Frank et al [11,12] using Raman spectrometry. While the free coordination sites in apo-TRF are loaded with Fe 3+ -ions, additional vibration modes from the coordination partners emerge in the Raman Spectra of the protein.…”

“…Figure 2. Selected Raman spectra from calibration solutions for iron saturation from 0.0 mol/mol (red) up to 1.0 mol/mol (blue) after data pretreatment and predicted versus measured plot from cross validation of the calibration model (inset) [11].…”