Abstract
Quantification of intact proteins in serum by liquid chromatography high-resolution mass spectrometry (HRMS) may be a useful alternative to bottom-up LC-MS or conventional ligand binding assays, due to reduced assay complexity and by providing additional information, such as isoform differentiation or detection of post-translational modifications. The 47.2 kDa lung cancer tumor marker neuron-specific enolase γ (NSEγ) was quantified in a clinically relevant concentration range of 6.25 to 100 ng/mL in NSE-depleted human serum using magnetic bead immunoprecipitation coupled to LC-high-resolution quadrupole-time-of-flight MS. The novelty of the described approach is in the combined setup of immunoaffinity extraction and the use of a full-length NSEγ calibrator and labeled NSEγ internal standard (IS) to reliably quantify the post-translationally acetylated form of this protein tumor marker in a top-down proteomics workflow. Isolation parameters and quantification using deconvolution and reconstructed extracted ion chromatograms were evaluated, and the development of a suitable liquid chromatography method was demonstrated. Various validation parameters were determined using both quantification methods, both showing acceptable performance. Additionally, deconvolution-based quantification enabled an accurate mass determination. The developed method was compared to a commercially available ECLIA and showed good correlation in sera of patients suspected of lung cancer. This assay may form the starting point for the development of a reference method for the standardization of immunoassays.
Original language | English |
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Pages (from-to) | 516-525 |
Number of pages | 10 |
Journal | Analytical Chemistry |
Volume | 97 |
Issue number | 1 |
Early online date | 22 Dec 2024 |
DOIs | |
Publication status | Published - 14 Jan 2025 |
Funding
This work was supported by The Netherlands Organization for Scientific Research through Gravity program 024.001.035.
Funders | Funder number |
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Nederlandse Organisatie voor Wetenschappelijk Onderzoek | 024.001.035 |
Keywords
- Humans
- Phosphopyruvate Hydratase/blood
- Biomarkers, Tumor/blood
- Mass Spectrometry/methods
- Lung Neoplasms/blood
- Chromatography, Liquid/methods