Evaluation of Comprehensive Quantification Assays for Plasma Protein Analysis Using a Novel Triple Quadrupole LC/MS

Importance of the Topic

Quantitative analysis of plasma proteins is foundational for biomarker discovery, clinical diagnostics, and translational research. Plasma represents a highly complex biological matrix with protein concentrations spanning multiple orders of magnitude. Advanced multiplexed assays are essential to achieve both sensitivity and reproducibility when profiling low-abundance proteins in such challenging samples.

Study Objectives and Overview

This study assessed a novel Agilent 6495 triple quadrupole LC/MS system for large-panel peptide quantification in human and mouse plasma. The goals were to compare its performance against a predecessor instrument, determine improvements in signal response and sensitivity, and demonstrate high-throughput capability for hundreds of targets in long-term runs.

Methodology and Instrumentation

• LC System: Agilent 1290 Infinity II Bio LC with ZORBAX RRHD Eclipse Plus C18 column (2.1×150 mm, 1.8 μm particles).
• Mass Spectrometer: Agilent 6495D Triple Quadrupole with 4th generation iFunnel and AJS ion source operating in positive mode.
• Gradient: 2% to 80% acetonitrile over a 50 min program at 0.4 mL/min, 10 µL injection, column temperature controlled.
• Acquisition: dynamic MRM (dMRM) methods configured in MassHunter 12.1 and Skyline for up to 2 250 transitions, sub-millisecond dwell times, and 650 ms cycle time.
• Samples: Stable isotope-labeled standard (SIS) peptides spiked into human or mouse plasma digests. Human plasma from Bioreclamation; PeptiQuant kits from MRM Proteomics.

Key Results and Discussion

• Enhanced Signal Response: The 6495D delivered a 2–3-fold increase in total MRM response compared to its predecessor.
• Sensitivity and Linearity: Quantification of a model SIS peptide showed linearity (R2 = 0.9996) from 1 amol/µL to 50 fmol/µL, with precision (CV < 12%) and accuracy within ±20% at the lowest levels.
• Noise and Chromatography: Even at 10 amol on-column, signal-to-noise ratio exceeded 150, demonstrating clear peak shapes in complex plasma digests.
• Large-Panel Performance: A 375-protein panel in mouse plasma used 2 250 transitions with 204 concurrent MRMs. Over 80% of peptides had dwell times <4 ms, and QC injections (n=9) showed median CV of 4.9% (100% of peptides CV < 20%) over three days.

Benefits and Practical Applications

• High Sensitivity: Detect low-abundance biomarkers in complex matrices with improved limits of detection and quantification.
• Robust Reproducibility: Stable peak areas and low CVs support longitudinal studies and large cohort analyses.
• Throughput and Multiplexing: Sub-millisecond dwell times and high concurrent transition capacity enable broad proteome coverage within a single run.
• Workflow Efficiency: Integrated autotune and method optimization tools reduce manual setup time and facilitate routine deployment in QA/QC and research labs.

Future Trends and Opportunities

• Expanded Target Panels: Scaling to thousands of peptides per run through optimized dMRM scheduling and hardware advances.
• Automation and AI Integration: Automated sample preparation and data processing with machine learning for peak detection and quantitation refinement.
• Clinical Translation: Standardized assays for personalized medicine and regulatory compliance in diagnostic laboratories.
• Miniaturization and Portability: Development of compact triple quadrupole platforms for point-of-care or field applications.

Conclusion

The Agilent 6495D triple quadrupole LC/MS system demonstrates significant enhancements in signal response, quantification sensitivity, and multiplexing capability for plasma protein analysis. These improvements facilitate robust, high-throughput assays suitable for biomarker research and clinical studies.