Definitive Protein Sequencing by Top-Down MS with timsOmni and OmniScape

Importance of the topic

Top-down mass spectrometry (TD-MS) offers direct analysis of intact proteoforms and preserves sequence connectivity that is frequently lost in bottom-up workflows. This capability is critical for unambiguous protein sequence assignment, detection of N- and C-terminal variants, verification of recombinant constructs (e.g., affinity tags), and accurate localization of single-residue substitutions or PTMs. The report illustrates how TD-MS combined with advanced ion mobility-enabled instrumentation and dedicated data-analysis workflows can correct erroneous bottom-up-derived sequences and deliver definitive protein identification with complete sequence coverage.

Objectives and overview of the study

The primary objective was to re-examine a protein sequence originally inferred from bottom-up proteomics using top-down experiments on a timsOmni platform and to reconstruct a corrected sequence using OmniScape software. The workflow aimed to (1) identify inconsistencies between BU-inferred and TD-measured masses, (2) perform de novo sequence reconstruction from multiple fragmentation modes, (3) iteratively trim or revise terminal regions (clipping analysis), and (4) screen for and confirm single-residue substitutions and final proteoform validation to reach 100% sequence coverage.

Methods and experimental workflow

The subject was a ~9 kDa protein that had been dialyzed, lyophilized, and reconstituted for analysis at 4 µM in 50:49.5:0.5 H2O:ACN:FA. Offline infusion on a timsOmni platform used an Apollo II ion source at 3 µL/min. The 8+ precursor charge state was isolated for MS2 experiments. Multiple fragmentation modalities were employed to generate complementary fragment ion series: collision-induced dissociation variants (CCID, RCID, CID), electron-based methods (ECD, ECciD, EID), and in-source CID (isCID). Data were processed in OmniScape using sequential workflows: de novo sequencing, clipping analysis, PTM/Single-site Substitution Screening, and confirmation workflows applied across MS2 datasets.

Instrumentation used

• timsOmni mass spectrometry platform (ion mobility-enabled MS).
• Apollo II electrospray source operated at 3 µL/min for offline infusion.
• Fragmentation modes: CCID, RCID, MS2 CID, ECD, ECciD, EID, and isCID.
• Data analysis: OmniScape software with De Novo, PTM Screening, Clipping Analysis, and Confirmation workflows.

Main results and discussion

• Initial discrepancy: The intact mass measured by TD-MS was higher than the mass predicted from the BU-inferred sequence, and MS2 ECD confirmation against the BU sequence produced only random fragment matches, indicating a mis-assigned BU sequence.
• De novo reconstruction: MS2 ECD de novo analysis produced a top-scored sequence tag that included an N-terminal His-tag connected to the protein N-terminus and revealed a 12-amino-acid N-terminal region that was not present in the BU-derived sequence. An MS2 isCID-derived tag supported portions of the BU-derived C-terminal sequence but indicated the BU sequence was too long.
• Iterative revision: A multistep revision strategy—de novo sequence generation, clipping analysis to remove C-terminal over-length, and targeted single-residue screening—was applied. Two specific residue substitutions were identified (reported as K45Q and K59Q in the final revision), and the N-terminal His-tag and additional N-terminal residues were confirmed.
• Spectral confirmation: Fully annotated MS2 ECD and ECciD spectra showed complete fragment annotation matching the revised sequence. Application of the confirmation workflows across MS2 CID, EID, and ECciD datasets validated the corrected proteoform, with ECciD achieving 100% sequence coverage (SC = 100%).
• Outcome: The combination of multiple fragmentation methods and OmniScape workflows transformed an initial zero-to-low top-down coverage on the BU-derived sequence into a fully validated corrected proteoform with complete sequence coverage and isotope-distribution agreement for the precursor.

Benefits and practical applications of the method

• Definitive sequencing: TD-MS combined with de novo and targeted screening workflows can resolve incorrect bottom-up assignments and confirm exact proteoform sequences including affinity tags and terminal processing events.
• Proteoform characterization: The multi-fragmentation approach increases confidence in mapping single-residue substitutions and PTMs, important for biotherapeutic QA/QC and discovery of biologically relevant proteoforms.
• Streamlined validation: Integrated software workflows (de novo → clipping → substitution screening → confirmation) enable systematic, reproducible sequence reconstruction from complex TD datasets.
• Complementarity to bottom-up: TD-MS is particularly valuable when peptide-centric methods fail due to sequence scrambling, proteolytically inaccessible regions, or ambiguous peptide inference.

Future trends and potential applications

• Broader adoption of ion mobility-enabled TD platforms coupled with robust de novo algorithms will expand routine proteoform-level characterization in both research and quality control settings.
• Improved software automation and visualization for iterative workflows (clipping, substitution screening) will shorten analysis time and lower required expertise for definitive sequence assignments.
• Integration with orthogonal techniques (top-down LC-MS, native MS, cross-linking) and machine-learning-assisted spectral interpretation could increase throughput and accuracy for larger proteins and complex modifications.
• Commercialization of turnkey TD-MS solutions and standardization of TD workflows will enable regulatory uptake for biologics characterization and batch release testing.

Conclusion

Using a timsOmni platform and OmniScape analytical workflows, the study corrected a bottom-up-inferred protein sequence by de novo reconstruction, clipping analysis, and single-site substitution screening. The final proteoform included an N-terminal His-tag and a newly identified 12-residue N-terminal segment, correction of C-terminal over-length, and two residue substitutions (K45Q and K59Q). Multiple fragmentation modes and confirmation workflows achieved full sequence coverage (SC=100%), demonstrating the power of top-down MS for definitive protein sequencing and proteoform validation.

Reference

No external literature references were listed in the source document. The findings are based on experimental data and analysis described in the provided summary (timsOmni platform datasets processed in OmniScape).