Q-RAI Provides Rapid and Sensitive Data Independent Acquisition for Peptide Quantitation

Significance of the Topic

Data independent acquisition with high resolution mass spectrometry delivers comprehensive fragment information for all ions, ensuring robust analysis of low-abundance peptides and critical quality attributes in protein therapeutics.

Study Objectives and Overview

This study evaluates the performance of Quadrupole Resolved All Ions (Q-RAI), an untargeted DIA mode, for sensitive and selective quantification of oxidation and deamidation modifications in NISTmAb peptides.

Methodology

Sample Preparation:

• Reduction, alkylation, overnight tryptic digestion at 37 °C.
• Forced oxidation with 0–1% H₂O₂ and forced deamidation at pH 9 for up to 14 days before digestion.

LC/Q-TOF Conditions:

• AdvanceBio Peptide Mapping column (2.1 × 150 mm) with a 25 min gradient.
• Twelve Q-RAI windows spanning m/z 300–1489 (100 amu each, 1 amu overlap), collision energy optimized per window.
• MS1 acquisition at 8 Hz and MS/MS at 14 Hz.

Data Analysis:

• Spectrum Mill for library creation from DDA runs.
• Skyline with mProphet scoring for DIA processing and quantitative evaluation.

Instrumentation

Agilent 6546 LC/Q-TOF system with high resolution, extended dynamic range, and Q-RAI acquisition capabilities.

Main Results and Discussion

• Mass errors for precursor and fragment ions remained below 5 ppm, demonstrating high mass accuracy and stability.
• mProphet yielded low p and q values, confirming data quality and reliable peak identification.
• Fragment ion intensities scaled proportionally across oxidation levels, enabling precise quantitation of modifications.
• Sequence coverage exceeded 95%, supporting comprehensive peptide mapping.

Benefits and Practical Applications

Q-RAI DIA offers sensitive and selective monitoring of critical quality attributes such as oxidation and deamidation in biopharmaceuticals, with the ability to generate untargeted peptide maps and robust quantitation.

Future Trends and Applications

• Integration with advanced spectral libraries and machine learning for automated PTM analysis.
• Higher throughput via reduced window widths and faster acquisition rates.
• Cloud-based data processing and real-time quality control in biomanufacturing.
• Extension to additional post-translational modifications and complex biological matrices.

Conclusion

Q-RAI mode on a high-resolution LC/Q-TOF platform provides reliable, high-quality DIA data for quantifying low-level peptide modifications, enhancing analytical workflows for therapeutic protein characterization.

References

1. Pino LK et al. Mass Spectrom Rev. 2020;39(3):229–244.
2. Doerr A. Nat Methods. 2015;12:35.
3. Reiter L et al. Nat Methods. 2011;8:430–435.