In-Depth Versatile Characterization of Aspartic Acid Isomers via a Cyclic Ion Mobility Mass Spectrometry (cIMS)

Importance of the Topic

Mass spectrometry-based separation of isobaric peptide isomers like Asp and isoAsp residues is critical for biotherapeutic characterization due to impacts on drug efficacy and safety. Ion mobility methods provide conformational separation that is not accessible by mass analysis alone, improving analytical confidence in complex protein mixtures.

Objectives and Study Overview

This study demonstrates the capabilities of a cyclic ion mobility mass spectrometry (cIMS) platform for in-depth differentiation of aspartic acid isomers. Two model systems were investigated: synthetic beta amyloid 1–16 peptides and tryptic peptides from a trastuzumab heavy chain (TmAb HC T12). The aim was to resolve isomeric species, localize modification sites, and assess the system’s flexibility in multi-pass separations and fragmentation experiments.

Methodology and Instrumentation

• Sample preparation: Infusion of synthetic peptide standards (0.5 μM in 10 mM ammonium acetate) and LC separation of reduced, alkylated, tryptic TmAb HC digest.
• Ion mobility separation: Single- and multi-pass experiments (up to 20 passes) on the cyclic IMS device, including mobility “slicing” for targeted re-injection.
• Fragmentation strategies: Collision-induced dissociation (CID) on mobility-selected precursors and electron capture dissociation (ECD) for site-specific isomer identification.

Used Instrumentation

• Waters ACQUITY H-Class UPLC system
• Waters SELECT SERIES Cyclic Ion Mobility Mass Spectrometer
• DIA mode with ion mobility (HDMSE) and targeted HDMS/MS

Main Results and Discussion

• Multi-pass cIMS achieved baseline resolution of Asp/isoAsp beta amyloid 1–16 isomers after 15–20 cycles, whereas single-pass separation was insufficient.
• Mobility “slicing” enabled reinjection of selected isomers for MSn and confirmed conformational differences.
• CID fragment mobility patterns pinpointed the isomerization site at residue 7 in the amyloid peptide.
• For TmAb HC T12 peptides, ion mobility and CID analyses distinguished the isoAsp variant (P3) from native-like species (P1/P2), and ECD provided further localization of modification.

Benefits and Practical Applications

• Enhanced characterization of therapeutic peptides and proteins with post-translational or degradation-related modifications.
• Improved analytical workflows in quality control for biopharmaceutical development.
• Flexibility in selecting and fragmenting specific mobility-resolved species for detailed structural analysis.

Future Trends and Applications

Combining cyclic ion mobility with advanced fragmentation techniques promises deeper insights into protein isoforms and other subtle modifications. Ongoing investigations will expand this approach to additional biotherapeutic candidates and integrate real-time processing algorithms to further accelerate characterization workflows.

Conclusion

The cyclic IMS-MS platform offers robust and versatile separation of aspartic acid isomers in complex peptide mixtures. Multi-pass mobility, targeted MSn, and complementary fragmentation modalities collectively enable precise isomer differentiation and localization, supporting stringent quality assessment of biotherapeutics.

Reference

• Zheng X; Chem Commun (Camb). 2017. 53 (56): 7913-7916.
• Yang H; J Proteome Res. 2009. 8 (10): 4615-4621.