Peptide Quantification in Plasma Using the Agilent 6495 Triple Quadrupole LC/MS Coupled with the Agilent 1290 Infinity II LC System

Importance of the Topic

The accurate quantification of peptide biomarkers in human plasma is essential for translational research, clinical diagnostics, and drug development. Reliable measurement of low-abundance proteins enables early disease detection, therapeutic monitoring, and validation of candidate biomarkers in large sample cohorts. Robustness, throughput, and sensitivity are critical when handling hundreds to thousands of plasma samples in high-volume studies.

Study Objectives and Overview

This study evaluates the quantitative performance of a standard-flow LC/MS setup consisting of the Agilent 1290 Infinity II LC system coupled with the Agilent 6495 triple quadrupole mass spectrometer and Jet Stream ionization source. Using a commercially available stable isotope-labeled peptide mixture, the work aims to define limits of quantification, linear dynamic range, precision, accuracy, and retention time reproducibility for 99 signature peptides derived from 75 protein biomarkers in a human plasma matrix.

Methodology and Instrumentation

Sample Preparation:

• Human plasma diluted and denatured with trifluoroethanol, reduced (DTT), alkylated (iodoacetamide) and digested overnight with trypsin.
• Peptide digest dried, reconstituted at ~1 µg/µL, and spiked with stable isotope-labeled peptide standards at nine concentration levels (5 amol/µL to 100 fmol/µL).

Instrumentation:

• Agilent 1290 Infinity II LC system with high-speed pump, cooled multisampler, and thermostatted column compartment.
• Agilent ZORBAX RRHD Eclipse Plus C18 column (2.1 × 150 mm, 1.8 µm) at 50 °C, flow rate 0.4 mL/min, 43 min gradient.
• Agilent Jet Stream ionization source, positive mode, optimized gas temperatures and flows.
• Agilent 6495C triple quadrupole mass spectrometer operated in dynamic MRM mode with ~600 transitions, unit resolution, 600 ms cycle time.

Main Results and Discussion

Chromatographic Performance:

• Sharp peak shapes across retention times from 2.4 to 36.5 min, median peak width (FWHM) 3.5 s.
• Retention time reproducibility excellent with RSD median of 0.13% over 45 injections.

Quantitative Figures of Merit:

• Linear dynamic range spanned five orders of magnitude for representative peptides (R² ≥ 0.99).
• Limits of quantification as low as 5 amol/µL in 1 µg/µL plasma digest for well-responding peptides.
• Precision at the LLOQ level ≤ 10% RSD; overall accuracy within 80–120% across concentrations.
• Matrix interference evaluated via qualifier/quantifier ion ratios; lowest reliable LLOQ set based on <20% bias.

Discussion:
The observed sensitivity matches or exceeds that of microflow systems while benefiting from higher throughput and simpler maintenance. Variation in peptide response underscores the need for careful transition selection. Dynamic MRM update and source optimization ensure retention accuracy over long gradients.

Benefits and Practical Applications of the Method

This standard-flow LC/TQ platform provides:

• High throughput suitable for large-scale biomarker verification.
• Robust operation with minimal drift in retention time and sensitivity.
• Sufficient analytical sensitivity to quantify low-abundance peptides in complex plasma matrices.
• Ease of method transfer and maintenance compared to low-flow systems.

Future Trends and Potential Applications

Advances may include integration of ion mobility for added selectivity, increased multiplexing through parallel reaction monitoring, and automation of sample preparation workflows. Expansion to non-tryptic peptides and post-translational modifications will broaden clinical applications. Cloud-based data analysis and AI-driven peak validation promise to streamline large cohort studies.

Conclusion

The Agilent 1290 Infinity II LC coupled to the 6495 triple quadrupole MS with Jet Stream ionization delivers outstanding performance for targeted peptide quantification in human plasma. The system achieves low-amol sensitivity, wide dynamic range, and high reproducibility, making it a compelling choice for high-throughput biomarker research.

Reference

• Agilent Technologies. The Agilent 6495 Triple Quadrupole LC/MS: Peptide Quantitation Performance. Technical overview, publication 5991-6898EN, 2016.
• Agilent Technologies. Jet Stream Proteomics for Sensitive and Robust Standard Flow LC/MS. Technical overview, publication 5991-5687EN, 2015.
• Percy AJ, et al. A Standardized Kit for Automated Quantitative Assessment of Candidate Protein Biomarkers in Human Plasma. Bioanalysis. 2015;7(23):2991–3004.
• Agilent Technologies. Robust and Reproducible Protein Quantification in Plasma using the Evosep One and the Agilent 6495 Triple Quadrupole LC/MS. Application note 5994-1928EN, 2020.