Electron Capture Dissociation in Combination With High Performance Cyclic Ion Mobility Separation for Detailed Protein Studies

Significance of the Topic

Electron Capture Dissociation (ECD) provides a nonergodic fragmentation pathway that preserves labile modifications and yields extensive sequence information for peptides and proteins. Combined with high-resolution cyclic Ion Mobility Spectrometry (IMS), ECD enables clear differentiation of isomeric species and enhanced top-down analysis, addressing critical challenges in proteomics and biopharmaceutical characterization.

Objectives and Overview

This study demonstrates the integration of ECD with a cyclic IMS-equipped quadrupole time-of-flight mass spectrometer (SELECT SERIES™ Cyclic™ IMS) to (1) separate and sequence isomeric phosphopeptides with high mobility resolving power and (2) apply pre-IMS ECD for top-down sequencing of bovine ubiquitin, exploiting IMS separation to boost sequence coverage and fragment identification.

Methodology

Samples included a 1:1 mixture of isomeric phosphopeptides (RSpYpSRSR and RYpSpSRSR) and the 10+ charge state of bovine ubiquitin. Cyclic IMS separations were performed with up to 25 passes (∼325 CCS/ΔCCS resolving power). ECD fragmentation was applied either post-IMS for isomer sequencing or pre-IMS for top-down analysis. Fragment ions were recorded by a high-resolution TOF analyzer and correlated to arrival times.

Used Instrumentation

• SELECT SERIES™ Cyclic™ IMS Mass Spectrometer (quadrupole TOF)
• Optional Electron Capture Dissociation (ECD) cell (pre- or post-IMS placement)
• Dual collision cells for CID (pre- and post-IMS)

Key Results and Discussion

Post-IMS ECD separated the phosphopeptides into two mobility distributions at ~208 ms and ~210 ms, enabling full sequence coverage and unambiguous assignment of phosphorylation sites. Pre-IMS ECD of ubiquitin yielded an IMS-resolved fragment ion map that simplified spectral interpretation. Mobility filtering doubled the number of detected fragments (110 to 253) and increased sequence coverage from 89% to 97%, enhancing assignment confidence.

Benefits and Practical Applications

• Site-specific characterization of labile modifications such as phosphorylation
• Unambiguous identification of isomeric peptides through high-resolution IMS
• Improved top-down protein coverage and fragment redundancy
• Versatile fragmentation strategies for fundamental research and biotherapeutic quality control

Future Trends and Opportunities

Advances may include integration of IMS-ECD with high-throughput proteomics and glycoproteomics, development of novel fragmentation modes (e.g., ultraviolet photodissociation), and application of machine learning for data interpretation. Continued improvements in IMS resolving power and instrument duty cycle will further expand detailed protein analysis capabilities.

Conclusion

The combination of ECD with high-performance cyclic IMS on a quadrupole TOF platform offers unprecedented ability to separate isomeric species and enhance top-down protein sequencing. This flexible approach supports both academic proteomics research and rigorous characterization of biotherapeutics.

References

1. Zubarev RA, Kelleher NL, McLafferty FW. Electron Capture Dissociation of Multiply Charged Protein Cations. A Nonergodic Process. J Am Chem Soc. 1998;120(13):3265–3266.
2. Bakhtiar R, Guan Z. Electron Capture Dissociation Mass Spectrometry in Characterization of Peptides and Proteins. Biotechnol Lett. 2006;28(14):1047–1059.
3. Shaw JB, Cooper-Shepherd DA, Hewitt D, et al. Enhanced Top-Down Protein Characterization with Electron Capture Dissociation and Cyclic Ion Mobility Spectrometry. Anal Chem. 2022;94(9):3888–3896.
4. Ippoliti S, Cooper-Shepherd DA, Yu Y-Q, Langridge JI. Confident O-glycosylation Site Identification for ENBREL (etanercept) Using the ECD Functionality of the SELECT SERIES Cyclic IMS System. Waters Application Brief. 2021.
5. Fellers RT, Greer JB, Early BP, et al. ProSight Lite: Graphical Software to Analyze Top-Down Mass Spectrometry Data. Proteomics. 2014;(15):1235–1238.