In-depth peptide mapping of biopharmaceuticals using an electron-transfer/higher-energy collision dissociation (EThcD) implemented on a modified Orbitrap hybrid MS

Significance of the Topic

Peptide mapping is a critical step in biopharmaceutical characterization. The ability to achieve complete sequence coverage, identify post-translational modifications (PTMs), and distinguish isomeric amino acids in a single LC-MS analysis enhances both research throughput and product quality control.

Objectives and Study Overview

This study aims to develop an in-depth peptide mapping workflow using electron-transfer/higher-energy collision dissociation (EThcD) on the modified Orbitrap Excedion Pro BioPharma mass spectrometer. The focus is on achieving:

• High sequence coverage of a monoclonal antibody digest
• Detection of low-abundance PTMs
• Unambiguous differentiation of isomeric amino acid residues

Methodology

The NISTmAb standard was denatured, reduced, alkylated, and digested with trypsin. The peptide digest was separated using a UHPLC gradient on a Hypersil Gold Peptide column and analyzed via LC-MS/MS. EThcD fragmentation parameters were optimized to maximize backbone cleavage while preserving labile modifications.

Instrumentation Used

• Orbitrap Excedion Pro BioPharma mass spectrometer configured for EThcD
• Thermo Scientific Vanquish Horizon UHPLC system
• Hypersil Gold Peptide UHPLC column (2.1×150 mm, 1.9 µm)

Main Results and Discussion

The EThcD workflow delivered 100% sequence coverage in a single run of NISTmAb digest. Ultra-low abundant deamidation (0.026 %) was confidently identified and quantified. Diagnostic w-type ions (z-43 and z-29) enabled clear differentiation of leucine and isoleucine, while c+57 and z-57 secondary fragments distinguished isoaspartic acid from aspartic acid even at 0.18 % abundance. High sensitivity was maintained across multiple charge states.

Benefits and Practical Applications

• Comprehensive characterization of therapeutic proteins
• Accurate localization of labile PTMs without over-fragmentation
• Reliable differentiation of isomeric residues critical for quality control and structure verification

Future Trends and Potential Applications

EThcD on advanced Orbitrap platforms may be extended to complex glycoprotein analysis, real-time process monitoring, and integration with data-independent acquisition strategies. Enhanced software algorithms will further improve PTM quantitation and isomer discrimination in high-throughput settings.

Conclusion

The implementation of EThcD on the Orbitrap Excedion Pro achieves unprecedented depth in peptide mapping. It combines high sensitivity, full sequence coverage, and the ability to resolve PTMs and isomeric amino acids in a single LC-MS run, making it a powerful technique for biopharmaceutical analysis.

References

1. Kjeldsen F. et al. Distinguishing Ile/Leu Amino Acid Residues in the PP3 Protein by (Hot) Electron Capture Dissociation in FT-ICR MS. Anal. Chem. 2003;75:1267–1274
2. Hurtado P.P. et al. Differentiation of isomeric amino acid residues in proteins and peptides using mass spectrometry. Mass Spectrometry Reviews. 2012;31:609–625