A Rapid and Economical Workflow for Protein Biomarker Discovery Using Agilent 6495D Triple Quadrupole LC/MS System

Importance of the topic

Protein biomarkers in human plasma are essential for early disease detection, therapy monitoring and personalized medicine. Developing a rapid and cost-effective workflow to discover and quantify these biomarkers accelerates research in clinical proteomics and quality control in biopharmaceutical manufacturing.

Goals and Study Overview

This proof-of-concept study aimed to establish a streamlined method for protein biomarker discovery in human plasma without relying on expensive stable-isotope standards. Key objectives included:

• Leveraging public peptide MS/MS spectral libraries and Uniprot human proteome data.
• Using Skyline software with the Agilent automation plugin for MRM method development.
• Evaluating assay performance in a cohort of 40 healthy subjects (20 male, 20 female).

Methodology

Sample Preparation:

• Human plasma samples underwent overnight trypsin digestion.
• Peptide cleanup was performed on the AssayMAP Bravo platform.
• A pooled QC sample was generated by combining aliquots of all digests.

MRM Method Development:

• 581 target proteins imported into Skyline using PeptideAtlas and Uniprot filters.
• Automated retention time (RT) prediction, collision energy optimization and transition selection completed within two days.
• Final dynamic MRM (dMRM) method comprised 3604 transitions covering 846 peptides from 390 proteins.

LC/MS Analysis:

• Agilent 1290 Infinity II Bio LC with ZORBAX RRHD Eclipse Plus C18 column.
• 25-minute gradient at 0.4 mL/min (0.1% formic acid in water and acetonitrile).
• Agilent 6495D Triple Quadrupole MS in positive AJS mode using dMRM acquisition.

Used Instrumentation

• Agilent 1290 Infinity II Bio LC system (high-speed pump, multisampler, multicolumn thermostat).
• Agilent 6495D Triple Quadrupole LC/MS system.
• AssayMAP Bravo peptide cleanup platform.
• MassHunter software v12.1 and Skyline-daily v23.1 with Agilent Automation plugin.

Main Results and Discussion

• RT Prediction Accuracy: 68% of endogenous peptides had RTs matching SIL-based measurements (R²=0.81).
• Quantitative Precision: Median coefficient of variation (CV) of QC peptide signals was 5.8%, with 91.7% of peptides below 20% CV.
• RT Reproducibility: Median RT CV across the study was 0.12%.
• Cohort Comparison: Four proteins showed significant gender‐related differences; pregnancy zone protein (PZP) peptides exhibited synchronized expression profiles.

Benefits and Practical Applications of the Method

• Rapid assay development in under two days without stable-isotope standards.
• Cost-effective workflow by omitting SIL peptides.
• High sensitivity with 846 peptides quantified in a single 25-min LC/MS/MS run.
• Robust reproducibility suitable for large cohort studies and routine QC.

Future Trends and Potential Applications

• Expansion to larger clinical cohorts and diverse biofluids.
• Integration with data-independent acquisition (DIA) for deeper proteome coverage.
• Automation of sample preparation and data processing for high-throughput screening.
• Adapting the workflow for regulatory-compliant diagnostics and biopharma quality control.

Conclusion

This study demonstrates a rapid, economical and reproducible workflow for protein biomarker discovery in human plasma using the Agilent 6495D LC/TQ system and Skyline software. By eliminating the need for stable-isotope standards and leveraging public spectral libraries, researchers can accelerate biomarker identification and quantification in clinical and industrial settings.

References

1. Gaither C et al. Determination of the concentration range for 267 proteins from 21 lots of commercial human plasma using highly multiplexed multiple reaction monitoring mass spectrometry. Analyst. 2020