MSACL 2026 Abstract

Optimization of Nano-LC–MRM-MS Enables High-Throughput Quantitative Serum Proteomics in Large Cardiovascular Disease Cohort

Kari Arnarson (1-4), Finnur F. Eiriksson (1,4), Valborg Gudmundsdottir (2,3), Christoph H Borchers (5-8), Vilmundur G. Gudnason (2,3), Margret Thorsteinsdottir (1,4,9).
(1) Faculty of Pharmaceutical Sciences, University of Iceland, Reykjavik, Iceland (2) Faculty of Medicine, University of Iceland, Reykjavik, Iceland (3) Icelandic Heart Association, Reykjavík, Iceland (4) ArcticMass, Reykjavik, Iceland (5) Segal Cancer Proteomics Centre, Lady Davis Institute for Medical Research, McGill University, Montreal, Quebec, Canada (6) Gerald Bronfman Department of Oncology, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, QC H3T 1E2, Canada (7) Division of Experimental Medicine, McGill University, Montreal, QC H4A 3J1, Canada (8) Department of Pathology, McGill University, Montreal, QC H3A 2B4, Canada (9) BioMedical Center, University of Iceland, Reykjavik, Iceland

Kári Arnarson, MSc (Presenter) University of Iceland

Presenter Bio: I am a PhD student at the Univeristy of Iceland, doing a joint project at the faculty of medicine and faculty of pharmaceutical sciences. My BSc in pharmacy was at the Univeristy of Iceland, and my MSc was split between the Univeristy of Iceland, Københavns Universitet, Copenhagen Denmark and a traineeship at the Center for Proteomics and Metabolomics at Leids Universitair Medisch Centrum, Leiden Netherlands.

Focus of my work is targeted mass spectrometry methods for clinical research. Specifically, my project is aimed at assay development for absolute quantification of protein biomarkers for cardiovascular disease.

Relevant Financial Disclosures (within past 24 months, reported on Apr 22, 2026) No relevant financial relationship(s) to disclose.

INTRODUCTION:
Cardiovascular disease remains the leading cause of non-communicable disease worldwide, underscoring the high unmet clinical need for predictive circulating protein biomarkers. Liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC–MRM MS) using stable isotope–labeled standard (SIS) peptides is a well established approach for absolute protein quantification in complex biological matrices. However, translation to large-scale clinical cohorts is constrained by limited sample throughput, high consumable costs, and extensive method optimization requirements. Systematic approaches to method development such as design of experiments (DoE), as well as advances in nano-LC provide opportunities to enhance analytical efficiency and cost-effectiveness without compromising quantitative performance.

OBJECTIVES:
This study aims to develop a high throughput nano LC–MRM MS platform for absolute quantitation of 32 circulating proteins, and to apply this platform to assess associations with myocardial infarction (MI) in a prevalent atherosclerotic cohort in Iceland.

METHODS:
A nano-LC–MRM MS platform was developed, and DoE strategy was used to optimize; digestion time, temperature, enzyme-to-substrate ratio and chaotropic agent (1). The platform targets 32 proteotypic peptides for 32 proteins previously associated with MI. For absolute quantification of the peptides, we utilized a customized MRM proteomics kit containing paired high purity unlabeled and SIS peptides for each target analyte. Quantitation was performed on an Evosep Eno nano LC system coupled to a Waters Xevo TQ Absolute triple quadrupole mass spectrometer operated in MRM mode. Analysis was performed on serum samples from 1,600 Icelandic individuals with prevalent atherosclerosis, collected across three sampling periods, with up to 18 years of follow up. Inclusion criteria for atherosclerotic burden were confirmed by carotid ultrasound (2).

RESULTS:
The analytical platform was successfully implemented, with DoE-guided optimization of sample preparation streamlining the workflow and reducing digestion time from overnight to 2 hours, without compromising quantitation accuracy or reliability. Sample handling was further simplified by direct loading of digested samples onto Evotip Pure tips, eliminating the need for offline solid-phase extraction and lyophilization. This combined optimization enables same-day sample preparation and analysis. Implementation of the Evosep Eno system reduced chromatographic run time from 60 to 12 minutes per sample while maintaining excellent retention-time reproducibility within and across analytical batches. Overall throughput increased tenfold while reducing reagent consumption by approximately tenfold, with analytical precision maintained at <20% relative standard deviation across all measured peptides. Correlation analyses between measured biomarkers and MI are ongoing.

CONCLUSION:
The reduction in time and SIS consumption enables scalability of the platform, representing an important step in toward clinical implementation of mass spectrometry, where turnover time and sample cost remain key barriers to accessibility.

REFERENCES:
1. Arnarson, K., Y. Holm, K., Gunnarsdottir Thormar, V., Eiríksson, F. F., Mohammed, Y., Borchers, C. H., Bodvarsdottir, S. K., & Thorsteinsdottir, M. (2025). Optimization of bottom-up proteomic sample preparation by design of experiments for absolute quantification of 257 proteins in human plasma using UPLC-MRM-MS. Expert Review of Proteomics, 1–8. https://doi.org/10.1080/14789450.2025.2504994
2. Sturlaugsdottir, R., Aspelund, T., Bjornsdottir, G., Sigurdsson, S., Thorsson, B., Eiriksdottir, G., & Gudnason, V. (2016). Prevalence and determinants of carotid plaque in the cross-sectional REFINE-Reykjavik study. BMJ Open, 6(11), e012457. https://doi.org/10.1136/bmjopen-2016-012457.