Top-Down Sequence Analysis of Intact Proteins Using an Agilent AdvanceBio 6545XT LC/Q-TOF with ExD

Importance of the topic

Proteoforms represent intact protein variants defined by unique amino acid sequences and post-translational modifications. Characterizing these species at the protein level is essential for understanding mechanisms of action in health and disease and for ensuring critical quality attributes in biotherapeutic development. Top-down mass spectrometry preserves proteoform-level information by fragmenting intact proteins, overcoming the limitations of peptide-level approaches that scramble proteoform context during digestion.

Objectives and study overview

This application note demonstrates a practical workflow for top-down sequence analysis of intact proteins using the Agilent AdvanceBio 6545XT LC/Q-TOF with an electron capture dissociation (ExD) cell. Key aims include guiding new users through instrument setup, exploring the effects of collision energy and spectral averaging on sequence coverage, and establishing a reproducible method for proteoform characterization across a range of protein sizes (8–46 kDa).

Methodology and instrumentation

An ExD-enabled 6545XT LC/Q-TOF was tuned for electron capture dissociation and combined with supplemental collision energy when needed. Proteins (ubiquitin, myoglobin, carbonic anhydrase, aldolase, enolase) were denatured, diluted in 15% acetonitrile/0.1% formic acid, and directly infused at 10–20 µL/min. Spectra were acquired at 1 spectrum/sec over m/z 200–3200 with a 9 m/z isolation window. Collision energy for CID experiments was optimized case by case, while ECD-only and ECD+CID methods were compared. Sequence deconvolution and fragment matching were performed using Agilent ExDViewer.

Main results and discussion

Electron capture dissociation alone achieved 100% sequence coverage for ubiquitin and 95% for myoglobin, far exceeding the best CID-only results (91% and 85% respectively). For larger proteins (29–46 kDa), supplementing ECD with low-level CID (10–40 V) increased unique fragment identifications and sequence coverage: carbonic anhydrase (62% vs. 41% CID-only), aldolase (38%), and enolase (23%). Spectral averaging improved signal-to-noise and coverage but exhibited diminishing returns beyond ~100 averaged spectra. Method reproducibility was confirmed over 52 days and between users, with a standard deviation of ±2.15% in sequence coverage for carbonic anhydrase.

Benefits and practical applications

• Preserves proteoform integrity and PTM correlations.
• Enables confident sequence confirmation across diverse protein sizes.
• Scalable direct-infusion workflow suitable for rapid QA/QC in biopharma.
• Transferable method with robust performance over time and between operators.

Future trends and opportunities

Advances in top-down instrumentation and software will drive broader adoption in proteoform profiling. Integrating ExD with other radical-driven dissociation methods and coupling with online separations promises higher throughput and deeper coverage. Application areas include biosimilar comparability, splice variant detection, PTM mapping in disease biomarker discovery, and quality control of complex biologics.

Conclusion

This work establishes a user-friendly, reproducible top-down MS workflow on an Agilent LC/Q-TOF platform, highlighting the synergistic use of ECD and CID for intact protein sequencing. The approach delivers high sequence coverage, reliable performance, and a scalable solution for proteoform analysis in research and biopharmaceutical settings.

References

1. Smith LM, Kelleher NL; The Consortium for Top-Down Proteomics. Proteoform: A Single Term Describing Protein Complexity. Nature Methods. 2013;10(3):186–187.
2. Smith LM, et al. The Human Proteoform Project: Defining the Human Proteome. Science Advances. 2021;7(50):eabk0734.
3. Keenan EK, et al. Discovering the Landscape of Protein Modifications. Molecular Cell. 2021;81(9):1868–1878.
4. Lutomski CA, et al. Multiple Roles of SARS-CoV-2 N Protein Facilitated by Proteoform-Specific Interactions. JACS Au. 2021;1(8):1147–1157.
5. Roberts DS, et al. Top-Down Proteomics. Nature Reviews Methods Primers. 2024;4:38.
6. Adams LM, et al. Mapping the KRAS Proteoform Landscape in Colorectal Cancer. Journal of Biological Chemistry. 2023;299(1):102768.
7. Fulcher JM, et al. Discovery of Proteoforms Associated with Alzheimer's Disease Through Quantitative Top-Down Proteomics. Molecular & Cellular Proteomics. 2024;24(6):100983.
8. Chapman EA, et al. Native Top-Down MS for Characterizing Sarcomeric Proteins Directly from Tissue. JASMS. 2024;35(4):738–745.
9. Beckman JS, et al. Improved Protein and PTM Characterization with Practical Electron-Based Fragmentation on Q-TOF. JASMS. 2021;32(8):2081–2091.
10. Gadkari VV, et al. Enhanced Collision Induced Unfolding and ECD of Native-Like Protein Ions. Anal Chem. 2020;92(23):15489–15496.