DATA INDEPENDENT TOP-DOWN MASS SPECTROMETRY FACILITATED BY A NEW MSE PROCESSING TOOL

Importance of Topic

A robust strategy for characterizing intact, post-translationally modified proteins is critical in biopharmaceutical research and quality control. Data-independent top-down mass spectrometry (TD-MSE) offers improved fragment ion signal for low-abundance species by fragmenting the entire charge state envelope. However, processing chromatographic TD-MSE data has been limited by coarse retention time window summation and high false-positive rates.

Objectives and Overview

This study describes a new software tool, TD-MSE, designed to process Synapt G2-Si TD-MSE data using advanced peak detection and apex alignment algorithms. The goals were to enhance sequence coverage and protein characterization scores (PCS) for low-abundance proteoforms, and to provide an interactive user interface supporting truncation searches, open modifications, and de novo tags.

Methodology and Instrumentation

The workflow employs a Waters Synapt G2-Si instrument with a 0.3×150 mm BioResolve intact protein column and a 15 min gradient (25–40 % ACN). Fragments are generated with a 20–30 V collision energy ramp in the TriWave trap, separated by ion mobility, then mass-analyzed. Software components include:

• IMTBX+grppr for deconvolution and two apex detection modes (FastApex, ApexFit)
• Python scripting and a Tkinter GUI
• Sliding sum and ScanByScan algorithms for chromatographic signal summation
• Options for peak matching, truncation±1000 Da, and open search for unspecified modifications

Main Results and Discussion

Applying a sliding 7-scan sum improved the signal-to-noise of low-abundance fragments, leading to increased sequence coverage of the Fc and Fc-Lys subunits of a degraded IdeS digest of NIST mAb. The sliding sum raised the number of internal ion identifications but also introduced false positives, reducing PCS when all fragments were counted. Manual removal of likely internal fragment hits restored high confidence scores. Peak alignment by apex rather than fixed retention windows doubled PCS relative to conventional MSE processing.

Benefits and Practical Applications

This approach:

• Enhances detection of low-abundance proteoforms in complex mixtures
• Improves sequence coverage through ensemble fragmentation
• Delivers higher confidence protein characterization scores
• Offers a flexible GUI for custom searches and rapid identification of missing termini

Future Trends and Applications

Advances may include expanded open-search capabilities for additional modifications, integration with alternative fragmentation methods (ECD, UVPD), and batch processing for high-throughput QC workflows. Further optimization of sliding-sum parameters and machine-learning–based peak scoring could reduce false positives while retaining enhanced coverage.

Conclusion

The TD-MSE processing tool demonstrates that combining sliding-sum signal aggregation with apex alignment and advanced peak fitting substantially improves fragment detection and confidence in TD-MSE experiments. This platform facilitates more complete characterization of therapeutic proteins and supports future developments in data-independent top-down proteomics.

References

1. Morrison L., Williams B., Sullivan B. Data Independent Top-Down Characterization of Proteins for Biotherapeutic Applications. ASMS Poster, 2016.
2. Avtonomov D.M., Polasky D.A., Ruotolo B.T., Nesvizhskii A.I. IMTBX and Grppr: Software for Top-Down Proteomics Utilizing Ion Mobility-Mass Spectrometry. Anal. Chem. 2018;90(3):2369–2375.