Immunoaffinity Intact Top-Down Mass Spectrometry for Quantification of Neuron-Specific Enolase Gamma, a Low-Abundance Protein Biomarker
Uloženo v:
| Název: | Immunoaffinity Intact Top-Down Mass Spectrometry for Quantification of Neuron-Specific Enolase Gamma, a Low-Abundance Protein Biomarker |
|---|---|
| Autoři: | Sebastian A. H. van den Wildenberg, Sylvia A. A. M. Genet, Maarten A. C. Broeren, Joost L. J. van Dongen, Maxime C.M. van den Oetelaar, Luc Brunsveld, Volkher Scharnhorst, Daan van de Kerkhof |
| Zdroj: | Anal Chem |
| Informace o vydavateli: | American Chemical Society (ACS), 2024. |
| Rok vydání: | 2024 |
| Témata: | Lung Neoplasms/blood, Chromatography, SDG 3 - Good Health and Well-being, Phosphopyruvate Hydratase/blood, Liquid/methods, Humans, Tumor/blood, SDG 3 – Goede gezondheid en welzijn, Biomarkers, Mass Spectrometry/methods |
| Popis: | 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. |
| Druh dokumentu: | Article Other literature type |
| Jazyk: | English |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/acs.analchem.4c04677 |
| Přístupová URL adresa: | https://pubmed.ncbi.nlm.nih.gov/39710932 |
| Rights: | CC BY URL: http://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (http://creativecommons.org/licenses/by/4.0/). |
| Přístupové číslo: | edsair.doi.dedup.....478316b2a23d09c0a6b4786165536758 |
| Databáze: | OpenAIRE |
Nájsť tento článok vo Web of Science | Abstrakt: | 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. |
|---|---|
| ISSN: | 15206882 00032700 |
| DOI: | 10.1021/acs.analchem.4c04677 |