Optimizing charge state distribution is a prerequisite for accurate protein biomarker quantification with LC-MS/MS, as illustrated by hepcidin measurement

Ellen M.H. Schmitz, Niels M. Leijten, Joost L.J. van Dongen, Maarten A.C. Broeren, Lech G. Milroy, Luc Brunsveld, Volkher Scharnhorst, Daan van de Kerkhof

Research output: Contribution to journalArticleAcademicpeer-review

5 Citations (Scopus)


Targeted quantification of protein biomarkers with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has great potential, but is still in its infancy. Therefore, we elucidated the influence of charge state distribution and matrix effects on accurate quantification, illustrated by the peptide hormone hepcidin. An LC-MS/MS assay for hepcidin, developed based on existing literature, was improved by using 5 mM ammonium formate buffer as mobile phase A and as an elution solution for solid phase extraction (SPE) to optimize the charge state distribution. After extensive analytical validation, focusing on interference and matrix effects, the clinical consequence of this method adjustment was studied by performing receiving operating characteristic (ROC)-curve analysis in patients with iron deficiency anemia (IDA, n=44), anemia of chronic disease (ACD, n=42) and non-anemic patients (n=93). By using a buffered solution during sample preparation and chromatography, the most abundant charge state was shifted from 4+ to 3+ and the charge state distribution was strongly stabilized. The matrix effects which occurred in the 4+ state were therefore avoided, eliminating bias in the low concentration range of hepcidin. Consequently, sensitivity, specificity and positive predictive value (PPV) for detection of IDA patients with the optimized assay (96%, 97%, 91%, respectively) were much better than for the original assay (73%, 70%, 44%, respectively). Fundamental improvements in LC-MS/MS assays greatly impact the accuracy of protein quantification. This is urgently required for improved diagnostic accuracy and clinical value, as illustrated by the validation of our hepcidin assay.

Original languageEnglish
Pages (from-to)1490-1497
Number of pages8
JournalClinical Chemistry and Laboratory Medicine
Issue number9
Publication statusPublished - 28 Aug 2018


  • charge state distribution
  • hepcidin
  • LC-MS/MS
  • mass spectrometry
  • protein quantification
  • Anemia, Iron-Deficiency/pathology
  • Area Under Curve
  • Biomarkers/analysis
  • Humans
  • Middle Aged
  • Male
  • Young Adult
  • Solid Phase Extraction
  • Aged, 80 and over
  • Adult
  • Female
  • Anemia/pathology
  • Tandem Mass Spectrometry/methods
  • C-Reactive Protein/analysis
  • Hepcidins/analysis
  • Chromatography, High Pressure Liquid/methods
  • Adolescent
  • ROC Curve
  • Aged
  • Chronic Disease


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