Molecular Hallmark Principles to Guide Protein and Metabolite Quantitation for Integrative Molecular Health Assessment

Molecular Hallmark Principles to Guide Protein and Metabolite Quantitation for Integrative Molecular Health Assessment Veronika Vidová1,2, Eliška Benešová1,2, Vojtěch Thon1, Gabriela Přibyl Dovrtělová1,2, Kapil Mandrah1, Hana Seličová1, Jana Klánová1,2 & Elliott James Price1,2 1 RECETOX, Faculty of Science, Masaryk University, Brno, Czech Republic 2 Czech node of the European Infrastructure for Human Exposome Research (EIRENE-CZ) Background •Limited assays for the absolute quantitation of extended sets of clinically-relevant protein & metabolite biomarkers. •We designed suite of biomarker assays for molecular health monitoring, each <50 µL of blood plasma/ serum, < 15 min runtime/ sample. •Clinical practitioners consulted for biomarker selection, contextualised around the Hallmarks of Health [1] and Hallmarks of Environmental Insult [2] frameworks. Example protein panel assay [3,4] Acknowledgements References This work was generated within the European Environmental Exposure Assessment Research Infrastructure (EIRENE RI). The work was supported by the EU’s H2020 research and innovation programme under grant agreement No. 857560 (CETOCOEN Excellence), by the HEU programme under grant agreement No. 101079789 (EIRENE PPP), by RECETOX Research Infrastructure (LM2023069) financed by the Ministry of Education, Youth and Sports, and Operational Programme Research, Development and Innovation – project CETOCOEN EXCELLENCE (No CZ.02.1.01/0.0/0.0/17_043/0009632) and from project SALVAGE (CZ.02.01.01/00/22_008/0004644) financed by MEYS – co-funded by the European Union. The views expressed are those of the author(s) and do not necessarily reflect those of the EU or REA. EJP is grateful for the MSACL 2025 Young Investigator Educational Grant. [1] López-Otín, C., & Kroemer, G. (2021). Hallmarks of Health. Cell, 184(1), 33–63. https://doi.org/10.1016/j.cell.2020.11.034. [2] Peters, A., Nawrot, T. S., & Baccarelli, A. A. (2021). Hallmarks of environmental insults. Cell, 184(6), 1455–1468. https://doi.org/10.1016/j.cell.2021.01.043. [3] Biomarker Analytical Laboratories. (2025). SOP for Quantification of inflammatory proteins in serum/plasma using UHPLC/SRM-MS. Zenodo. https://doi.org/10.5281/zenodo.14974161. [4] Biomarker Analytical Laboratories. (2025). Multiplex SRM assay validation. Panorama Public. https://doi.org/10.6069/hn2n-nv57 (plasma), https://doi.org/10.6069/x714-y552 (serum). [5] Benešová, E., Vidová, V., & Spáčil, Z. (2021). A comparative study of synthetic winged peptides for absolute protein quantification. Sci Rep. May 25;11(1):10880. https://doi.org/10.1038/s41598021-90087-9. [6] Benešová, E., Vidová, V., & Spáčil, Z. (2021). Synthetic winged peptides validation data. Panorama Public. https://doi.org/10.6069/q4ej-t746. Scheme of biomarker assay panels currently within the EIRENE-CZ node, with the LC-QQQ protein panel for inflammatory and immunity protein biomarkers as example case. Workflow of validated assay for the absolute quantification of 33 inflammatory and immunity proteins/ proteoform biomarkers [3,4]. •Proteins (proteoform) selection: IgA1, A1AT (3), SAA1, SAA2 (2), SAA4, CRP (2), A1AG1, A1AG2, ApoE, A2AP (2), CX3CL1, CHGA, ApoA1, IgG1, IgG2, IgG3, IgG4, HPβ, HPα2, A2M, CAL1, CAL2, MPO, CST3, AMBP, KNG1 (3), LBP, B2M. Example results evidencing unique individual IgG profiles, with identification of IgG2 and IgG4 deficiencies (patient Z) and elevated IgG4 (patients X & Y). •Quantification by double winged stable isotopically labelled peptides [5,6] and dynamic multiple reaction monitoring mass spectrometry. •Provides personalised results, including identification of clinically-relevant polymorphism and missense mutations.