Lecture
High‑Throughput Targeted Proteomics for Large‑Scale Quantification of Plasma Protein Biomarkers
- at -
- ICM Saal 2
- Type: Lecture
Lecture description
M. Thorsteinsdottir1, K. Arnarson1, V. Gudmundsdottir1, V. Gudnason1, C. Borchers2, F. Eiriksson1
¹ University of Iceland, Reykjavik, Iceland
² McGill University, Montreal, Canada
Absolute quantification of protein biomarkers using liquid chromatography–tandem mass spectrometry (LC–MRM‑MS) with stable isotope–labeled standard (SIS) peptides is a powerful approach for targeted proteomics, but its application in large clinical cohorts is often limited by low throughput and high reagent costs. We developed a high‑throughput nano‑LC–MRM‑MS workflow designed to increase analytical efficiency and reduce consumable use while maintaining robust quantitative performance.
A design‑of‑experiments strategy was applied to optimize key parameters of bottom‑up proteomic sample preparation, resulting in an improved protocol consisting of a 2‑hour trypsin digestion at 37 °C with a 1:5 (w:w) enzyme‑to‑protein ratio using guanidine hydrochloride as the denaturant. The optimized method was integrated with the Evosep Eno nano‑LC platform and Waters Xevo TQ Absolute mass spectrometer for MRM‑based quantification of 32 plasma proteins implicated in incident myocardial infarction.
Peptide separations were completed in 12 minutes per sample with excellent retention‑time reproducibility and improved chromatographic peak shapes across all analytes. SIS peptide usage was reduced ten‑fold compared with conventional UPLC–MRM‑MS while achieving comparable signal intensities. Sample handling was simplified by direct loading onto Evotip Pure tips, eliminating offline cleanup steps. Analytical validation demonstrated strong accuracy and precision (<20% RSD) across all measured peptides.
The assay was applied to longitudinal plasma samples from 200 participants collected over 20 years, enabling efficient analysis of ~10,000 samples at the cost previously required for ~1,000. Quantified protein levels correlated strongly with established cardiometabolic risk factors, confirming the platform’s suitability for large‑scale biomarker verification studies. This high‑throughput nano‑LC–MRM‑MS workflow substantially reduces cost and analysis time, offering a scalable solution for population‑level protein biomarker quantification.
¹ University of Iceland, Reykjavik, Iceland
² McGill University, Montreal, Canada
Absolute quantification of protein biomarkers using liquid chromatography–tandem mass spectrometry (LC–MRM‑MS) with stable isotope–labeled standard (SIS) peptides is a powerful approach for targeted proteomics, but its application in large clinical cohorts is often limited by low throughput and high reagent costs. We developed a high‑throughput nano‑LC–MRM‑MS workflow designed to increase analytical efficiency and reduce consumable use while maintaining robust quantitative performance.
A design‑of‑experiments strategy was applied to optimize key parameters of bottom‑up proteomic sample preparation, resulting in an improved protocol consisting of a 2‑hour trypsin digestion at 37 °C with a 1:5 (w:w) enzyme‑to‑protein ratio using guanidine hydrochloride as the denaturant. The optimized method was integrated with the Evosep Eno nano‑LC platform and Waters Xevo TQ Absolute mass spectrometer for MRM‑based quantification of 32 plasma proteins implicated in incident myocardial infarction.
Peptide separations were completed in 12 minutes per sample with excellent retention‑time reproducibility and improved chromatographic peak shapes across all analytes. SIS peptide usage was reduced ten‑fold compared with conventional UPLC–MRM‑MS while achieving comparable signal intensities. Sample handling was simplified by direct loading onto Evotip Pure tips, eliminating offline cleanup steps. Analytical validation demonstrated strong accuracy and precision (<20% RSD) across all measured peptides.
The assay was applied to longitudinal plasma samples from 200 participants collected over 20 years, enabling efficient analysis of ~10,000 samples at the cost previously required for ~1,000. Quantified protein levels correlated strongly with established cardiometabolic risk factors, confirming the platform’s suitability for large‑scale biomarker verification studies. This high‑throughput nano‑LC–MRM‑MS workflow substantially reduces cost and analysis time, offering a scalable solution for population‑level protein biomarker quantification.
