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

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 929; pp. 125 - 132
• Main Authors: Torsetnes, Silje Bøen, Løvbak, Sandra Gransbråten, Claus, Cecile, Lund, Hanne, Nordlund, Marianne S., Paus, Elisabeth, Halvorsen, Trine Grønhaug, Reubsaet, Léon
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.06.2013
• Subjects: accuracy; biomarkers; blood serum; chromatography; Chromatography, Liquid - methods; Humans; Immunocapture; Isoenzymes - blood; isozymes; lung neoplasms; Neuron-specific enolase; peptides; phosphopyruvate hydratase; Phosphopyruvate Hydratase - blood; Reproducibility of Results; Serum; Signature peptide; Tandem Mass Spectrometry - methods; Validation; Validation; Serum; Immunocapture; Signature peptide; Neuron-specific enolase
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2013.04.010

•Determination of cancer markers allow to understand pathological states.•Immunoextraction of the NSE's γ-subunit results in low complexity and high enrichment.•Tryptic digestion and LC-separation is performed prior to SRM-MS-detection.•This validated method differentiates isoenzymes of the cancer marker NSE through signature peptides for α- and γ-enolase.•NSE in serum, at both reference level and elevated levels, is quantified. 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 5–500ng/mL), intra-day precision <13% RSD, and accuracy >95%), and has a limit of quantification (of 38pg/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±0.45ng/mL of γ-subunit of NSE.