Simultaneous quantification of proteins in human serum via sulfur and iron using HPLC coupled to post-column isotope dilution mass spectrometry

Abstract

The quantification of proteins by inductively coupled plasma-mass spectrometry (ICP-MS) offers an alternative method for quantitative proteomics. In this study, we developed a method based on high performance liquid chromatography (HPLC) coupled to ICP-MS via post-column isotope dilution for the quantification of transferrin (Tf) and albumin (Alb) in human serum using enriched ³⁴S and ⁵⁴Fe isotopic solutions. First, Tf, lactoglobulin (Lacb), myoglobin (Myo), and lysozyme (Lys) serving as model proteins were separated with a size exclusion column and permitted the quantification of individual proteins by post-column addition of an enriched ³⁴S spike solution. In this way, the methodology was established and validated by comparing with gravimetric results. Further, after Tf saturation and by introducing isotopically enriched ³⁴S and ⁵⁴Fe spikes at the same time, the human serum Tf was absolutely quantified via both sulfur and iron by means of species-unspecific isotope dilution HPLC-ICP-MS. Good agreement of the two results from sulfur and iron were acquired. Moreover, the concentrations of Tf and Alb in human serum via sulfur using HPLC-ID-ICP-MS were simultaneously determined for the first time by altering the flow rate of enriched ³⁴S isotope solution. All the proposed species-unspecific HPLC-ID-ICP-MS methods were tested for the analysis of a serum certified reference material (ERM-DA470k/IFCC).