Analysis of Neuron–Specific enolase isozymes in human serum using immunoaffinity purification and liquid chromatography–tandem mass spectrometry quantification

Sylvia A.A.M. Genet, Jur R.E. Wolfs, Chris B.A.K. Vu, Madita Wolter, Maarten A.C. Broeren, Joost van Dongen, Luc Brunsveld, Volkher Scharnhorst, Daan van de Kerkhof (Corresponding author)

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Neuron–specific enolase (NSE) is a promising small–cell lung cancer (SCLC) biomarker composed of αγ and γγ isozyme dimers. As the conventional immunoassays are prone to interferences and cannot differentiate between the isozymes, we developed a multiplex immunoaffinity (IA) liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for the quantification of NSEα and NSEγ in human serum. A calibrator was prepared by performing cold denaturation of recombinantly expressed αα and γγ enolase dimers to induce a new dimer equilibrium that was determined to be approximately 1αγ:1γγ:1αα. Selective sample purification was achieved by performing IA extraction using an antibody specific towards NSEγ. The isolated αγ and γγ dimers were denatured and trypsin digested to allow quantification of the selected signature peptides and their corresponding isotopically labelled peptide internal standard. The obtained linear dynamic ranges were determined to be 1.5–56 ng/mL and 0.64–167 ng/mL for NSEα and NSEγ (R2 = 0.88 and 0.97 respectively). Validation of the assay showed acceptable accuracy and precision for NSEα and NSEγ. The method was successfully applied to patient serum in which both isozymes were detected. Compared to the conventional immunoassay, substantially lower total NSE concentrations were measured in IA LC–MS/MS. With this multiplex IA LC-MS/MS assay, the clinical value of quantifying the individual isozymes can be explored. In addition, together with the calibrator described here, it may be applied to standardize NSE immunoassays across different platforms.

Original languageEnglish
Article number123701
Number of pages7
JournalJournal of Chromatography, B, Analytical Technologies in the Biomedical and Life Sciences
Publication statusPublished - 15 May 2023

Bibliographical note

Funding Information:
The study was supported by The Netherlands Organization for Scientific Research (NWO) with LIFT grant 731.017.405. Roche Diagnostics partially sponsored the NSE reagents used in this study. The funding organizations did not play a role in the study design, data interpretation or preparation of approval of the paper.


  • immunoaffinity (IA)
  • Liquid chromatography–tandem mass spectrometry (LC–MS/MS)
  • Lung cancer
  • Neuron–specific enolase (NSE)
  • Reversible protein denaturation
  • Tryptic digestion
  • Chromatography, Liquid/methods
  • Reproducibility of Results
  • Peptides
  • Isoenzymes
  • Humans
  • Tandem Mass Spectrometry/methods
  • Phosphopyruvate Hydratase


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