Peptide Mapping of Trastuzumab Tryptic Digests on an Agilent 6545XT AdvanceBio LC/Q-TOF

Importance of the Topic

Peptide mapping by LC-MS/MS is essential for verifying monoclonal antibody primary sequence and for precise localization of PTMs such as deamidation and glycosylation in biopharmaceutical development and quality control. Its ability to combine MS1 and MS2 data ensures comprehensive molecular characterization of therapeutic proteins, supporting their efficacy, safety and regulatory compliance.

Objectives and Study Overview

The study aimed to establish a robust peptide mapping workflow for tryptic digests of trastuzumab using an Agilent 6545XT AdvanceBio LC/Q-TOF. Key goals included achieving high sequence coverage across replicates, demonstrating dynamic range for low-abundance peptides, and confidently identifying PTM sites including deamidation and glycoforms.

Methodology

Trastuzumab samples underwent reduction, alkylation and overnight digestion with Trypsin/LysC at a 25:1 protein-to-enzyme ratio in 1 M guanidine HCl buffer. No desalting was applied prior to LC/MS analysis. Chromatographic separation used an Agilent 1290 Infinity II Bio LC system with an AdvanceBio Peptide Mapping column (2.1 × 150 mm, 2.7 μm). The 6545XT AdvanceBio LC/Q-TOF was configured with dual Jet Stream ESI, optimized at a fragmentor voltage of 95 V to balance ion transmission and minimize in-source fragmentation. MS1 and data-dependent MS/MS were acquired at 3 spectra/s, covering m/z 200–3000 and 100–3000, respectively. Data processing employed Agilent MassHunter BioConfirm 12.1 with stringent filters (5 ppm MS1, 20 ppm MS2, BioScore ≥ 5).

Instrumentation Used

• Agilent 1290 Infinity II Bio LC system
• Agilent 6545XT AdvanceBio LC/Q-TOF with dual Jet Stream ESI
• AdvanceBio Peptide Mapping column, 2.1 × 150 mm, 2.7 μm
• MassHunter BioConfirm software, version 12.1

Main Results and Discussion

Sequence coverage for trastuzumab reached 98–99.7% across three replicates, with the only missing segment at the glycan site N300. Trypsin autolysis peptides accounted for ~54% coverage, highlighting the importance of monitoring enzyme fragments. Representative low-abundance peptides with MS1 signals near 1 × 10^4 exhibited complete fragment ladders, demonstrating extended dynamic range. Deamidation of peptide FTISADTSKNTAYLQMNSLR was unambiguously localized at Asn17 through characteristic b- and y-ion shifts. Glycopeptide mapping of TKPREEQYNSTYR revealed multiple glycoforms (G0, G0F, G1F, etc.) with MS1 intensities in the 10^5 range and confirmed peptide backbone fragments at m/z values above 1500 in MS/MS despite dominant glycan fragmentation.

Benefits and Practical Applications

• Confident confirmation of mAb primary structure with >98% coverage
• Precise localization of deamidation and other PTMs
• Robust glycoform profiling in a single workflow
• Sensitive detection of low-abundance peptides for quality control

Future Trends and Potential Applications

Anticipated advances include automation of sample preparation, integration of ion mobility for additional separation of isobaric PTMs, AI-driven spectral analysis to streamline data interpretation, and expansion of multiplexed workflows for simultaneous characterization of multiple biotherapeutics. Enhanced column chemistries and ultra-high-resolution mass analysis may further improve glycoform and PTM profiling depth.

Conclusion

The described peptide mapping approach on an Agilent 6545XT AdvanceBio LC/Q-TOF delivers high sequence coverage, dynamic range for low-abundance peptides, and reliable PTM localization. This workflow offers a comprehensive solution for monoclonal antibody characterization in research and regulated environments.

References

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