Comprehensive Protein Quantification in Plasma Using the Agilent 6495D Triple Quadrupole LC/MS System

Importance of the Topic

The accurate and sensitive quantification of protein biomarkers in human plasma is critical for biomarker validation, clinical research, and drug development. Multiplexed targeted proteomics using liquid chromatography–tandem mass spectrometry (LC–MS/MS) with multiple reaction monitoring (MRM) offers precise, reproducible measurements of low-abundance proteins in complex matrices. Advances in instrument sensitivity, automated method development, and high-throughput operation can accelerate biological insight and reduce method setup time.

Objectives and Overview of the Study

This application note assesses the performance of the Agilent 6495D triple quadrupole LC/MS system combined with an Agilent 1290 Infinity II Bio LC and MassHunter Workstation software version 12.1. The goals were to demonstrate ultrahigh analytical sensitivity, enhanced signal response, automated MRM method development via an integrated Skyline-Agilent workflow, and high precision quantification over a broad dynamic range of peptide standards in human plasma.

Methodology and Instrumentation

• Instrumentation:
  – Agilent 1290 Infinity II Bio LC system (high-speed pump, multisampler with thermostat, multicolumn thermostat)
  – Agilent 6495D triple quadrupole LC/MS (AJS source, iFunnel ion focusing, submillisecond dwell times)
  – Software: Agilent MassHunter Workstation v12.1 and Skyline from the MacCoss lab
• Chromatographic Conditions:
  – Column: ZORBAX RRHD Eclipse Plus C18, 2.1×150 mm, 1.8 µm
  – Mobile phases: water/0.1% formic acid (A), acetonitrile/0.1% formic acid (B)
  – Flow rate: 0.4 mL/min; injection: 10 µL; gradient: 2→30% B over 50 min, ramp to 80% B, total 55 min run + 5 min post time
• Sample Preparation:
  – Human plasma diluted in 25 mM ammonium bicarbonate, denatured with TFE, reduced (DTT), alkylated (iodoacetamide), digested with trypsin overnight
  – Stable isotope-labeled standard (SIS) peptides spiked to generate calibration standards (1.25 amol/µL to 50 fmol/µL in 0.5 µg/µL digest)
• MRM Method Development:
  – Automated Skyline-Agilent workflow for retention time determination and collision energy optimization
  – Exported dynamic MRM (dMRM) methods and static MRM panels without manual tuning

Main Results and Discussion

• Automated Workflow Integration:
  – MassHunter v12.1 integrates Skyline to generate optimized dMRM methods, reducing hands-on time and ensuring consistent method parameters
• Signal Response Gain:
  – Head-to-head comparison with predecessor instrument showed a median 2.4-fold increase in MRM signal across 101 peptide pairs in plasma
  – Example peptide (ATEHLSTLSEK) signal increased from ~764 to ~1,670 counts, with improved precision (RSD 8.8% vs 17.7%)
• Quantification Sensitivity and Linearity:
  – Achieved lower limit of quantification (LLOQ) at 1.25 amol/µL for selected SIS peptides (R2 > 0.99)
  – Dynamic range linearity tested from 1.25 amol/µL to 50 fmol/µL with accuracy within 80–120% and RSD < 20%
• High-Throughput Capability and Precision:
  – Static MRM panel of 303 concurrent transitions with 0.5 ms dwell time, 832 ms cycle time delivered ≥17 data points per 0.25 min peak
  – Twenty replicates of plasma digest showed 79 peptides with RSD <20% and 60 peptides with RSD <10%
• Retention Time and Response Reproducibility:
  – Four representative peptides exhibited RT RSD ≤0.3% and response RSD ≤9.6% across 20 injections

Benefits and Practical Applications of the Method

The combination of iFunnel-enhanced sensitivity, submillisecond dwell times, and AI-driven auto-tune/reflex workflows enables large-panel assays for hundreds of peptides in a single run. Researchers can rapidly implement standardized panels (up to 270 plasma biomarkers) or develop custom MRM assays with minimal manual intervention. High precision and low LLOQs support biomarker validation, clinical proteomics, and quality control in biopharmaceutical laboratories.

Future Trends and Applications

Continued integration of AI-based tuning and predictive maintenance will further streamline method setup and instrument uptime. Expanding multiplexing capacity and automating data-driven reflex decisions will enhance throughput. Applications may extend to personalized medicine, longitudinal biomarker monitoring, and integration with high-resolution MS workflows for enhanced proteome coverage.

Conclusion

The Agilent 6495D triple quadrupole LC/MS system coupled with the 1290 Infinity II Bio LC and MassHunter v12.1 demonstrates ultrahigh sensitivity, robust precision, and automated workflow capabilities for comprehensive protein quantification in plasma. This platform facilitates rapid assay deployment, broad dynamic range quantification, and supports large-scale targeted proteomics studies.

References

1. Mohammed Y, et al. Absolute Quantitative Targeted Proteomics Assays for Plasma Proteins. Methods Mol. Biol. 2023, 2628, 439–473.
2. 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, 145(10), 3634–3644.
3. Triple Quadrupole LC/MS Peptide Quantitation with Skyline Workflow Guide B.08.02. Agilent Technologies, publication 5990-9887EN, 2018.