Quantification of human serum transferrin using liquid chromatography–tandem mass spectrometry based targeted proteomics

Currently, the absolute quantification of human transferrin (hTRF) is based on several techniques other than mass spectrometry. Although these techniques provide valuable information on protein levels and can be extremely sensitive, they often lack the specificity and reproducibility that can be provided by mass spectrometry. In this study, a liquid chromatography–tandem mass spectrometry (LC/MS/MS) based targeted proteomics assay was developed and validated for the determination of transferrin in human serum. We selected the tryptic peptide 108EDPQTFYYAVAVVK121 as the surrogate analyte for quantification and used a stable isotope-labeled synthetic peptide with this sequence as an internal standard. Sample cleanup and enrichment were achieved using solid phase extraction. The validated calibration range was from 500 to 5000 ng/mL. The intra- and inter-day precisions were less than 4.9% and 9.0%, respectively. The bias for the quality control (QC) samples was less than 5.4%. Finally, this assay was successfully applied to the quantitative analysis of transferrin in clinical samples. The obtained values were assessed by independently measuring transferrin in the same samples using a commercially available immunoturbidimetric assay. As a result, the absolute concentrations determined by the LC/MS/MS assay compared well with those obtained with the immunoturbidimetric method; however, the LC/MS/MS assay afforded more reliable transferrin values at low concentrations.

► A novel LC/MS/MS-based targeted proteomics assay was developed and validated.
► Protein digestion efficiency and surrogate peptide selection were discussed.
► The LC/MS/MS assay was compared with a commercial immunoturbidimetric method.
► Clinical monitoring of prospective protein biomarkers could be achieved by LC/MS/MS.