A modified SLIM-IM-QTOF for high resolution collision induced unfolding and native protein analysis

Significance of the topic

The structural interrogation of intact proteins and protein complexes under native conditions is critical for biopharmaceutical characterization, quality control, and fundamental structural biology. Collision induced unfolding (CIU) coupled to high‐resolution ion mobility‐mass spectrometry (IM‐MS) provides detailed insights into conformational stability, domain organization, and ligand interactions without extensive sample preparation or labeling.

Objectives and study overview

• To modify a commercially available SLIM‐IM‐QTOF (MOBIE® 1.0 HRIM‐QTOF) with in‐source ion activation hardware for high‐resolution CIU experiments.
• To evaluate the performance of the modified instrument using model proteins (BSA, HSA) and a panel of monoclonal, bispecific, and trispecific antibodies.
• To compare unfolding fingerprints and reproducibility against a benchmark Agilent 6560C IM‐QTOF.

Methodology and instrumentation

Proteins were buffer‐exchanged into 200 mM ammonium acetate and infused via a micro‐nebulizer and syringe pump at 5–10 µM. In‐source collision energy (0–435 V ramp) induced progressive unfolding of charge states of interest. Mobility separation was performed on a 13 m SLIM device, and mass analysis on a QTOF. Data processing and CIU fingerprint analysis were conducted in CIUSuite 2.

Instrumental setup

• SLIM‐IM‐QTOF (MOBIE® 1.0 HRIM‐QTOF) equipped with in‐source collision activation hardware upstream of the SLIM path.
• Agilent 6560C IM‐QTOF for performance comparison, using identical CIU protocols.

Key results and discussion

• Modified HRIM‐QTOF produced high‐resolution CIU fingerprints for BSA/HSA charge states +17 and +18 with RMSD ~ 12–13 %, closely matching 6560C results and demonstrating reproducibility.
• Monoclonal IgG1κ, IgG2κ, IgG1λ and IgG2λ displayed distinguishable CIU50 values and unfolding transitions; cross‐comparison RMSD values ranged from 3 % to 19 %, enabling antibody variant screening by CIU profile.
• Bispecific and trispecific antibody pairs showed low intra‐pair RMSD (3–4 %) but significant inter‐pair differences (up to 14 %), indicating sensitivity to hinge region modifications and overall architecture.
• F(ab’)2 fragments of Herceptin and NIST mAb yielded distinct CIU fingerprints (RMSD > 10 %), whereas intact forms were indistinguishable, highlighting domain‐specific analysis advantages.

Benefits and practical applications

• High‐resolution CIU on a modified SLIM platform enables detailed stability profiling of antibodies and protein complexes.
• Rapid screening of biosimilars, antibody variants, and engineered constructs without extensive sample manipulation.
• Enhanced separation of conformers and more defined unfolding transitions improves analytical confidence.

Future trends and potential applications

Advances may include integration with top‐down fragmentation for proteoform mapping, automated high‐throughput CIU screening workflows, and expansion to large protein assemblies. Improved ion activation control and SLIM path modifications may further boost resolution and sensitivity for complex biological assemblies.

Conclusion

The incorporation of in‐source activation into a SLIM‐IM‐QTOF platform successfully enabled high‐resolution CIU under native conditions. The modified instrument matched the performance of established IM‐QTOF systems, offering reliable, reproducible unfolding fingerprints for proteins and antibody constructs. This approach provides a powerful tool for structural screening and quality assessment in biopharmaceutical research.

References

• Gadkari VV, et al. Analyst. 2023;148(2):391–401.
• Kurulugama RT, et al. ASMS 2022, TP 298; ASMS 2023, ThP 395.
• Polasky DA, et al. Anal Chem. 2019;91(4):3147–3155.