Quantification of the lung cancer tumor marker CYFRA 21-1 using protein precipitation, immunoaffinity bottom-up LC-MS/MS

Abstract

Objectives Numerous studies have proven the potential of cytokeratin 19 fragment 21-1 (CYFRA 21-1) detection in the (early) diagnosis and treatment monitoring of non-small cell lung cancer (NSCLC). Conventional immunoassays for CYFRA 21-1 quantification are however prone to interferences and lack diagnostic sensitivity and standardization. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an emerging approach based on a different, often superior, detection principle, which may improve the clinical applicability of CYFRA 21-1 in cancer diagnostics. Therefore, we developed and validated a protein precipitation, immunoaffinity (IA) LC-MS/MS assay for quantitative analysis of serum CYFRA 21-1. Methods Selective sample preparation was performed using ammonium sulfate (AS) precipitation, IA purification, tryptic digestion and LC-MS/MS quantification using a signature peptide and isotopically labeled internal standard. The workflow was optimized and validated according to EMA guidelines and results were compared to a conventional immunoassay. Results Significant interference effects were seen during IA purification, which were sufficiently solved by performing AS precipitation prior to IA purification. A linear calibration curve was obtained in the range of 1.0–100 ng/mL (R2=0.98). Accuracy and precision were well within acceptance criteria. In sera of patients suspected of lung cancer, the method showed good correlation with the immunoassay. Conclusions A robust AS precipitation-IA LC-MS/MS assay for the quantification of serum CYFRA 21-1 was developed. With this assay, the clinically added value of LC-MS/MS-based detection over immunoassays can be further explored.

Immunoaffinity Intact-Mass Spectrometry for the Detection of Endogenous Concentrations of the Acetylated Protein Tumor Biomarker Neuron Specific Enolase

“…Antibody coupling to protein G functionalized magnetic Dynabeads was performed as published previously. 17 Briefly, 2 μg of anti-human NSE 9601 SPTN-5 monoclonal mouse antibody (LOT: 0046841, Medix Biochemica, Espoo, Finland) (anti-NSEγ antibody) was coupled to 0.25 mg of Protein G Dynabeads (Thermo Fisher Scientific, Waltham, MA, USA) to obtain one equivalent, suitable for one isolation experiment. BS(PEG) 5 (Sigma-Aldrich, Saint Louis, MO, USA) crosslinking of the protein G and antibody was performed at a concentration of 25 μM.…”