In-Depth Protein Sequence Verification by TIMS-enabled MALDI Top-Down Sequencing

Importance of the topic

Trapped ion mobility separation combined with MALDI in-source decay top-down sequencing offers a robust approach for detailed protein characterization. By isolating fragment ion charge states, this hybrid method enhances sequence confirmation and de novo analysis, addressing key challenges in proteoform assignment and antibody variable region identification.

Objectives and Study Overview

• Demonstrate TIMS-enabled MALDI-TDS for high-coverage sequence confirmation
• Compare performance on carbonic anhydrase II and NISTmAb subunits (Fc/2, LC, Fd)
• Evaluate de novo sequencing capability using mobility-separated 1+ and 2+ fragments and pseudo-MS³ (T³-Sequencing)
• Assess homology search strategies in public and custom databases

Methodology and Instrumentation

• Sample preparation: Bovine carbonic anhydrase II and IdeS-digested NISTmAb subunits separated by LC and dried onto MALDI target in sDHB matrix
• Data acquisition: MALDI-ISD TIMS-MS on a timsTOF fleX with red phosphorus calibration
• Data processing: TIMS extraction, smoothing and baseline correction in DataAnalysis 6.1
• Sequence analysis: Bruker OmniScape for confirmation and de novo tag generation, online MS-BLAST and local NovorTag searches against SWISSPROT plus NISTmAb entries

Main Results and Discussion

• Near 100 % sequence coverage achieved for all NISTmAb subunits by combining mobility-separated 1+ and 2+ MALDI-ISD fragments and T³-Sequencing
• Charge purification via TIMS improved detection of low-abundance 2+ fragments, enhancing core region readout
• Offline LC-MALDI decoupling delivered high-resolution, accurate spectra without chromatographic time constraints
• Variable domain CDRs 1 and 2 fully covered; CDR3 coverage reached ~80 % with 2+ fragment contribution
• Fc/2 sequence confirmed against public database, while LC and Fd required custom NISTmAb-augmented searches for precise identification

Benefits and Practical Applications

• Comprehensive proteoform verification in biopharmaceutical QA/QC workflows
• Accurate de novo sequencing for proteins lacking complete reference entries
• Improved antibody variable region analysis critical for biosimilar development and characterization
• Flexibility of offline LC-MALDI for complex sample separation and high-quality data acquisition

Future Trends and Potential Applications

• Integration of higher-order MSn on TIMS platforms to resolve remaining sequence gaps
• Expansion of custom database searches with machine-learning-driven tag assembly
• Application to other complex proteoforms, post-translational modification mapping and neoantigen discovery
• Automation of workflow for high-throughput antibody screening and validation

Conclusion

Combining TIMS separation with MALDI-ISD top-down sequencing and advanced software tools yields near-complete protein sequence coverage in a single experiment. The approach enhances detection of multiple charge states, supports accurate de novo assembly, and streamlines homology searches in both public and custom databases. This methodology represents a significant advancement for detailed proteoform and antibody characterization.

Reference

• D. Suckau, A. Resemann (2003) T³-Sequencing targeted characterization of the N- and C-termini of undigested proteins by mass spectrometry Anal Chem 75(21)
• A. Shevchenko et al. (2001) Charting the proteomes of organisms with unsequenced genomes by MALDI-quadrupole time-of-flight mass spectrometry and BLAST homology searching Anal Chem 73(9)