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

Neuron-specific enolase (NSE) is a promising small-cell lung cancer (SCLC) biomarker composed of alpha gamma and gamma gamma 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 NSE alpha and NSE gamma in human serum. A calibrator was prepared by performing cold denaturation of recombinantly expressed alpha alpha and gamma gamma enolase dimers to induce a new dimer equilibrium that was determined to be approximately 1 alpha gamma:1 gamma gamma:1 alpha alpha. Selective sample purification was achieved by performing IA extraction using an antibody specific towards NSE gamma. The isolated alpha gamma and gamma gamma 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 NSE alpha and NSE gamma (R2 = 0.88 and 0.97 respectively). Validation of the assay showed acceptable accuracy and precision for NSE alpha and NSE gamma. 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.