Accurate Determination of Human Serum Transferrin Isoforms: Exploring Metal-Specific Isotope Dilution Analysis as a Quantitative Proteomic Tool

Abstract

Carbohydrate-deficient transferrin (CDT) measurements are considered a reliable marker for chronic alcohol consumption, and its use is becoming extensive in forensic medicine. However, CDT is not a single molecular entity but refers to a group of sialic acid-deficient transferrin isoforms from mono- to trisialotransferrin. Thus, the development of methods to analyze accurately and precisely individual transferrin isoforms in biological fluids such as serum is of increasing importance. The present work illustrates the use of ICPMS isotope dilution analysis for the quantification of transferrin isoforms once saturated with iron and separated by anion exchange chromatography (Mono Q 5/50) using a mobile phase consisting of a gradient of ammonium acetate (0−250 mM) in 25 mM Tris-acetic acid (pH 6.5). Species-specific and species-unspecific spikes have been explored. In the first part of the study, the use of postcolumn addition of a solution of 200 ng mL^-1 isotopically enriched iron (⁵⁷Fe, 95%) in 25 mM sodium citrate/citric acid (pH 4) permitted the quantification of individual sialoforms of transferrin (from S₂ to S₅) in human serum samples of healthy individuals as well as alcoholic patients. Second, the species-specific spike method was performed by synthesizing an isotopically enriched standard of saturated transferrin (saturated with ⁵⁷Fe). The characterization of the spike was performed by postcolumn reverse isotope dilution analysis (this is, by postcolumn addition of a solution of 200 ng mL^-1 natural iron in sodium citrate/citric acid of pH 4). Also, the stability of the transferrin spike was tested during one week with negligible species transformation. Finally, the enriched transferrin was used to quantify the individual isoforms in the same serum samples obtaining results comparative to those of postcolumn isotope dilution and to those previously published in the literature, demonstrating the suitability of both strategies for quantitative transferrin isoform determination in real samples.