MSⁿ Characterization of Chemical Modifications in Pharmaceutical Protein Byproducts Using the timsOmni Platform

Significance of the topic

Targeted structural characterization of low-abundance chemical modifications in pharmaceutical protein byproducts is essential for ensuring drug stability, efficacy and safety. Small proteins and heterogeneous proteoforms frequently carry subtle mass changes (e.g., deamidation, oxidation, sulfur additions) that are difficult to distinguish by intact-mass (MS1) alone. High-confidence, site-specific assignments are required to elucidate degradation pathways, support quality control and regulatory documentation, and guide formulation or process mitigation strategies.

Study objectives and overview

The study demonstrates an LC-MSn workflow on the timsOmni platform designed to resolve ambiguous MS1 assignments and localize low-level chemical modifications in synthetic pharmaceutical protein byproducts. The work compares complementary ion-activation methods (collision- and electron-based) for targeted MS2/MSn interrogation of modified proteoforms, evaluates data-processing strategies for PTM screening and localization, and highlights cases where MS2 corrected misassignments from MS1 interpretation.

Methods and used instrumentation

• Sample type: Small synthetic pharmaceutical proteins (minibinder protein A and a second protein B exhibiting pyroglutamylation and an additional +32 Da adduct).
• Chromatography: Reversed-phase LC using an Elute+ system (Bruker) for online separation and targeted selection of LC peaks.
• Mass spectrometry platform: timsOmni (Bruker) with Omnitrap ion handling, enabling dynamic ion accumulation and controlled electron irradiation.
• Ion activation strategies: A combination of complementary dissociation methods was applied on a per-target basis, including collision-induced dissociation (CID/cCID), electron capture dissociation (ECD), electron-induced dissociation (EID) and hybrid ECciD approaches.
• Data acquisition: Targeted LC-timescale MSn workflows to isolate and fragment selected chromatographic peaks for in-depth proteoform interrogation.
• Data processing: OmniScape software for systematic PTM screening, fragment annotation, localization scoring and mass error analysis.

Main results and discussion

• Protein A (minibinder): Two distinct LC peaks were observed corresponding to different deamidation products. Targeted MS2 using ECD, EID, CID and ECciD provided complementary fragment ion series that allowed localization of one deamidation to a C-terminal asparagine (higher coverage and multiple supporting fragments) and revealed a mixture of two closely spaced N-terminal deamidation proteoforms for the second peak. Electron-based methods contributed diagnostic c- and z-type ions that increased confidence relative to CID alone.
• Protein B: An observed +32 Da adduct initially annotated as dioxidation from MS1 data was re-evaluated after targeted MS2 analysis. Systematic mass error shifts (ppm) were observed for fragment ions that contained two oxygens when annotated as dioxidation. Reassigning the 32 Da shift as a sulfur addition consistent with a trisulfide bridge reconciled the mass accuracy across fragments and confirmed trisulfide formation as the true modification. This case underlines the potential for MS1-only assignments to misidentify chemically similar mass shifts and the value of MS2-based validation.
• Overall, combining collision-based and electron-based dissociation furnished orthogonal fragment information: CID provided abundant b/y ions, while ExD methods yielded complementary c/z and radical-driven fragments that helped pinpoint modification sites and distinguish isobaric or near-isobaric chemistries.

Benefits and practical applications

• Improved confidence in PTM assignments at low abundance by leveraging targeted MSn on the LC timescale.
• Capability to discriminate chemically similar mass shifts (e.g., oxidation vs. sulfur-containing adducts) that could otherwise mislead intact-mass interpretation.
• Site-specific localization of modifications to inform degradation pathway analysis, formulation development, and quality-control decision-making.
• Methodological flexibility: complementary CID + ExD strategies enable broader sequence coverage and diagnostic ion types useful for regulatory submissions and root-cause investigations.

Future trends and potential applications

• Broader adoption of hybrid MSn workflows combining ion-mobility-enabled separations, dynamic ion trapping and controlled electron irradiation to increase sensitivity and specificity for low-level proteoforms.
• Integration of advanced software tools for automated PTM screening, mass-error pattern recognition and confidence scoring to accelerate identification and reduce analyst bias.
• Application to more complex biotherapeutics (e.g., larger proteins, multisubunit assemblies, ADCs) and to real formulation/degradation samples to expand the method’s practical impact.
• Development of standardized MSn-based strategies for regulatory guidance, enabling harmonized approaches to characterize critical quality attributes and degradation products.

Conclusion

Targeted LC-MSn using complementary collision- and electron-based activation on the timsOmni platform provides a robust approach to structurally characterize low-level chemical modifications in pharmaceutical protein byproducts. The combined workflow increases site localization confidence, corrects misassignments that arise from MS1-based interpretation, and supports practical needs in biopharma quality control and degradation analysis. The demonstrated capability to distinguish dioxidation-like mass shifts from sulfur-containing modifications (e.g., trisulfide bridges) exemplifies the analytical value of MS2-driven validation.

References

1. K.F. Haselmann, M.E. Kraner, H. Jensen, T.N. Vinther, A. Smyrnakis, M. Kosmopoulou, K. Winkels, D. Baluya, C. Albers & D. Papanastasiou. MSn Characterization of Chemical Modifications in Pharmaceutical Protein Byproducts Using the timsOmni Platform. ASMS 2026. Affiliations include Novo Nordisk, Fasmatech, Bruker Daltonics and Bruker Scientific.