Targeted Peptide Quantification with Small Foot-print Capillary LC-MS/MS

Significance of the Topic

Quantifying peptides derived from low-abundance proteins is essential in proteomics research and biopharma quality control. Traditional standard-flow LC-MS/MS often lacks the sensitivity and requires larger sample and solvent volumes. Capillary LC-MS/MS addresses these limitations by enhancing detection limits, reducing sample requirements, and lowering solvent and gas consumption.

Objectives and Study Overview

This work aimed to establish a robust capillary LC-MS/MS workflow for targeted quantification of host cell protein (HCP) peptides in monoclonal antibody (mAb) digests. Key goals included:

• Determining limits of detection (LOD) and quantification (LOQ)
• Evaluating linear dynamic range
• Assessing precision and accuracy
• Comparing sensitivity with standard-flow LC-MS/MS

Methodology and Instrumentation

Sample Preparation:
The IgG1 mAb was produced in CHO cells, purified, and digested with trypsin following denaturation, reduction, and alkylation. Heavy stable isotope-labeled (SIL) peptide standards were spiked at seven concentrations (62.5–50 000 amol) into the digest for calibration curves.

LC-MS/MS Analysis:
Axcend Focus capillary LC was interfaced with an Agilent Ultivo Triple Quadrupole LC/MS operating in multiple reaction monitoring (MRM) mode. A five-minute reversed-phase gradient on a 150 µm × 100 mm C18 column (1.8 µm) at 2 µL/min was used. Agilent Automation tools, Skyline, and MassHunter optimized MRM transitions automatically.

Instrumentation Used

• Axcend Focus Capillary LC System
• Agilent Ultivo Triple Quadrupole LC/MS
• Electrospray Ionization (ESI) Source
• Agilent Jet Stream (AJS) Ion Source
• Agilent MassHunter Workstation Software
• Skyline Software for MRM Optimization

Main Results and Discussion

Sensitivity Improvement:
Capillary LC-MS/MS achieved LODs as low as 62.5 amol for all four HCP peptides, compared with 125–1000 amol for standard-flow methods. ESI mode on the capillary system delivered up to 10-fold higher peak intensities, and AJS mode gave a 2-fold gain over standard-flow.

Solvent and Gas Savings:
The capillary setup consumed ~250-fold less organic solvent and ~4-fold less nebulizer and drying gas than standard-flow LC/MS.

Linearity and Dynamic Range:
Calibration curves for all peptides displayed linearity coefficients (R2) > 0.998 across nearly three orders of magnitude (62.5–50 000 amol).

Precision and Accuracy:
Intra-run precision (%RSD) was generally below 10%, and accuracy ranged between 88% and 112% over the concentration range. Retention time reproducibility remained within 1% RSD.

Benefits and Practical Applications of the Method

• High sensitivity suitable for trace-level HCP analysis in biopharma workflows
• Minimal sample volume requirements for precious or scarce materials
• Rapid five-minute gradients enabling higher throughput
• Substantial cost and environmental benefits through reduced solvent and gas use

Future Trends and Opportunities

Further development may focus on:

• Expanding multiplexed MRM methods for larger peptide panels
• Integration with microfluidic sample preparation to minimize handling losses
• Applications to post-translational modification analysis at low abundance
• Automation and real-time data processing for high-throughput screening

Conclusion

This capillary LC-MS/MS approach provides a sensitive, precise, and efficient workflow for targeted peptide quantification. The method significantly outperforms standard-flow LC/MS in detection limits, dynamic range, and resource consumption, offering a practical solution for proteomics and biopharmaceutical quality control.

References

Agilent Technologies, Inc. and Axcend. Poster WP 587, ASMS 2024.