Species-Specific Isotope Dilution Methodology for the SI Traceable Quantification of Serum Human Ferritin Light Chain (FTL)

There is an urgent need for reference methodologies to assign SI traceable values to quality control (QC) materials for protein biomarkers to validate biochemical tests currently used in disease diagnosis and patient-specific therapeutics. Here we describe a novel methodology specifically developed for the SI traceable quantification of human serum ferritin light chain (FTL), an established biomarker for the diagnosis of iron-related disorders such as hemochromatosis. Natural and ³⁴S isotopically enriched human FTL standards were recombinantly expressed using the HEK-293 mammalian cell system. Both standards were fully characterized for their total sulfur content, isotopic abundance, species distribution and primary sequence before using them for isotope dilution analysis (IDA). Treatment of serum spiked with FTL was optimized to selectively remove high abundant serum proteins without affecting significantly the integrity of the FTL. This was achieved by using methanol protein precipitation in combination with heat treatment and ultrafiltration. Complementary asymmetrical field-flow-fractionation (AF4) and anion-exchange high performance liquid chromatography (AE-HPLC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) were used to monitor the impact of sample treatment on protein removal and FTL recovery. Both standards, natural and enriched, were used with the optimal sample treatment in a double species-specific isotope dilution analysis (SS-IDA) workflow for the determination of FTL in the WHO "4th International Standard for Ferritin" (NIBSC 19/118) resulting in a protein mass fraction of 11.0 with an expanded uncertainty (U, <i>k</i> = 2) of 1.0 μg mL^-1 (RSU of 9.4%). The method was validated by standard addition experiments, and a full uncertainty budget was established. For the first time sulfur SS-IDA has been used to assign an SI traceable value to a relatively low abundance serum protein biomarker at supraphysiological concentrations in a WHO standard. This methodology will be invaluable for adding SI traceability to existing QC materials and for the certification of new biomarker reference materials.