CE/MS/MS as an Orthogonal Technique for Sensitive and Easy Quantification of Peptides in Complex Matrixes

Importance of the Topic

Accurate quantification of peptides in complex biological samples underpins proteomics research, clinical diagnostics and biopharmaceutical quality control. Capillary electrophoresis coupled to tandem mass spectrometry (CE/MS/MS) offers an orthogonal separation technique that enhances sensitivity and selectivity, especially for low-abundance peptides in challenging matrices.

Objectives and Overview

This study demonstrates a CE/MS/MS workflow for sensitive, high-confidence quantification of two human serum albumin (HSA) peptides spiked into an Escherichia coli tryptic digest. The work highlights the integration of Agilent’s 7100 CE system with a 6490 triple quadrupole mass spectrometer using the Agilent Jet Stream (AJS) triple tube sprayer and unified MassHunter software for seamless control and data acquisition.

Methodology and Instrumentation

Sample Preparation:

• Synthetic HSA peptides (YLYEIAR and LVNEVTEFAK) prepared at 20 pmol/µL in water and serially diluted to 0.002–20 pmol/µL.
• Spiking of peptides into E. coli tryptic digest for complex matrix evaluation.

Separation Conditions:

• Capillary: 60 cm × 50 µm i.d. fused silica; buffer: 1 M acetic acid, pH 2.3; injection: 10 s at 50 mbar; voltage: 30 kV; temperature: 20 °C.
• Sheath liquid: 1:1 methanol/water with 0.5% acetic acid at 8 µL/min supplied by an isocratic pump with flow splitter.

Mass Spectrometry:

• Agilent 6490 Triple Quadrupole MS in positive AJS mode; MRM acquisition.
• Optimized qualifier and quantifier transitions via MassHunter Peptide Optimizer; dynamic MRM potential noted.
• MRM transitions imported from Skyline for expanded multiplexing (e.g., 60 transitions for BSA digest).

Results and Discussion

The two HSA peptides migrated closely at 3.8 min (YLYEIAR) and 3.9 min (LVNEVTEFAK) under acidic CE conditions, demonstrating baseline resolution of peptides with similar charge states. Calibration curves over four orders of magnitude (0.002–20 pmol/µL) yielded R2 > 0.999. Limits of detection and quantitation were 2.3 amol and 23.5 amol, respectively, even in the E. coli digest background. Overlay of 60 BSA tryptic peptide transitions confirmed the system’s capability for high-throughput, multiplexed quantification in a single 35 min run.

Benefits and Practical Applications

• Orthogonal separation enhances peptide coverage and confidence compared to LC/MS alone.
• Attomole sensitivity enables trace-level quantification in complex biological and biopharmaceutical samples.
• Unified software control simplifies switching between CE/MS and LC/MS platforms, streamlining workflows in research and QA/QC laboratories.

Future Trends and Opportunities

Advances may include dynamic MRM scheduling for improved cycle time, coupling CE/MS with high-resolution mass analyzers for deeper proteome coverage, and expanding applications to clinical glycoproteomics, biomarker validation and real-time process monitoring in biomanufacturing.

Conclusion

This application note establishes CE/MS/MS as a robust, highly sensitive and user-friendly strategy for peptide quantification in complex matrices, offering a valuable complement to conventional LC/MS workflows.

Reference

1. Sun L. et al. Analyst. 2013;138:3181–3188.
2. Glycopeptide Analysis of Antibodies by Capillary Electrophoresis and Q-TOF MS, Agilent 5990-7138EN.
3. Compatibility of Agilent Jet Stream with CE/MS, Agilent 5990-9716EN.
4. MacLean B. et al. Bioinformatics. 2010;26(7):966–968.