Immunocapture and LC–MS/MS for selective quantification and differentiation of the isozymes of the biomarker neuron-specific enolase in serum

NSE, neuron-specific enolase, is an important biomarker for several pathological conditions including small cell lung cancer (SCLC). The current paper presents an LC–MS/MS-based approach for quantification of NSE in serum at both reference levels and elevated levels. The analytical approach utilizes selective sample preparation by immunoextraction of all forms of NSE (αγ, γγ, and γ) followed by tryptic digestion, and separation and detection by LC–SRM-MS. The quantification of NSE is performed through a signature peptide specific for the γ-subunit of NSE (tryptic peptide γ16; ELPLYR). The method is validated and shows satisfactory results (linearity r2 > 0.999 (range 5–500 ng/mL), intra-day precision <13% RSD, and accuracy >95%), and has a limit of quantification (of 38 pg/mL; S/N = 10) significantly lower than endogenous levels of healthy subjects. In addition, the method simultaneously allows determination of the αγ-heterodimer through a signature peptide specific for the α-subunit (tryptic peptide α12; TIAPALVSK). The method was successfully applied to serum samples from healthy blood donors. In all samples from healthy blood donors both the α- and the γ-subunit was detected (S/N > 200 for both signature peptides), confirming the presence of the αγ-heterodimer in these sample. The level in one of them was determined to be (n = 5) 7.3 ± 0.45 ng/mL of γ-subunit of NSE.