Robust and Reproducible Protein Quantification in Plasma using Evosep One and Agilent 6495 Triple Quadrupole LC/MS

Importance of the Topic

The accurate quantification of low-abundance protein biomarkers in plasma is critical for clinical research and diagnostic applications. Robust, high-throughput LC/MS workflows that deliver consistent retention times, stable signal responses, and low limits of quantification across large sample sets are essential to support biomarker discovery and validation.

Objectives and Study Overview

This study evaluated the performance of a low-flow proteomics platform combining Evosep One chromatography with an Agilent 6495 triple quadrupole mass spectrometer. Goals included assessing retention time reproducibility, signal stability over hundreds of injections, and analytical sensitivity for targeted peptides in human plasma.

Used Instrumentation

• Evosep One LC system with disposable preformed gradient trap columns
• Agilent Nanospray source (G1992A) with stainless-steel emitter
• Agilent 6495 Triple Quadrupole LC/MS operating in dynamic MRM mode monitoring 33 peptide pairs

Methodology

Human plasma was denatured, reduced, alkylated, and trypsin-digested, then dried and reconstituted. A stable isotope-labeled peptide mixture was spiked into the digest at varying concentrations for calibration. Samples (~1 µg digest) were loaded onto Evotips and separated using a 21-minute preformed gradient on an 8 cm × 100 µm C18 column at 1 µL/min. The mass spectrometer monitored 33 pairs of heavy and endogenous peptides matching 31 protein biomarkers in dynamic MRM mode.

Main Results and Discussion

• Retention times for all targeted peptides remained highly reproducible over 574 consecutive injections (RSD 0.43–2.75%).
• MRM peak area median RSD was 8.5%, with 94% of peptides below 16% RSD, demonstrating stable signal response.
• Four exemplar peptides showed signal RSD of 6.0–7.9% and retention time RSD of 0.59–1.04% across all injections without instrument adjustments.
• Standard curves for the SIS peptide SGIPIVTSPYQIHFTK achieved an LLOQ of 10 amol and an LOD of 4 amol on-column, both before and after the robustness test, confirming sustained sensitivity.

Benefits and Practical Applications

This workflow enables high-throughput, reproducible quantification of plasma protein biomarkers with minimal maintenance. Disposable trap columns and preformed gradients simplify sample loading and reduce carryover, making the approach suitable for large clinical cohorts and routine QA/QC in pharmaceutical and biomarker laboratories.

Future Trends and Opportunities

Emerging developments may include even shorter gradients for greater throughput, expanded MRM panels for larger biomarker coverage, and integration with automated data analysis pipelines. Applications could extend to personalized medicine, longitudinal patient monitoring, and rapid therapeutic response assessments.

Conclusion

The combination of Evosep One low-flow LC with the Agilent 6495 triple quadrupole mass spectrometer provides a robust, reproducible, and sensitive platform for high-throughput plasma proteomics, ensuring reliable biomarker quantification across extensive sample series.