Improvement of throughput and charge assignment accuracy in Orbitrap-based charge detection mass spectrometry with proton transfer charge reduction

Improving Orbitrap-based Charge Detection Mass Spectrometry (CDMS) Through Proton Transfer Charge Reduction (PTCR)

Significance of the topic

Charge detection mass spectrometry (CDMS) directly measures m/z and the integer charge of single ions, enabling unambiguous mass determination for large, heterogeneous biomolecules. However, CDMS throughput and charge-assignment reliability are limited when analyte ions occupy a narrow, congested low m/z region: Automatic Ion Control (AIC) limits how many ions can be sampled per acquisition to avoid multi-ion events, and charge misassignments produce large mass errors. Integrating gas-phase charge-reduction chemistry (PTCR) prior to Orbitrap CDMS can redistribute ions to higher m/z, reduce spectral congestion, and thereby improve sampling efficiency, sensitivity for high-mass fragments, and charge-determination accuracy. These improvements are important for top-down proteomics, native MS, and analysis of intact macromolecular assemblies where heterogeneity and high m/z complexity are common.

Objectives and study overview

• Demonstrate that PTCR applied before Orbitrap-based CDMS increases the number of ions sampled per spectrum and raises the fraction of confidently charge-assigned ions.
• Quantify gains in detection of high-mass fragment ions and resultant improvements in top-down proteoform sequence coverage using bovine carbonic anhydrase II (bCA II) as a model.
• Assess how PTCR affects charge-assignment algorithms, with emphasis on the central-limit method used for unresolved isotope distributions.

Methodology and instrumentation

• Sample: Bovine carbonic anhydrase II (bCA II) prepared by buffer exchange into 200 µM ammonium acetate and diluted 100-fold from a 1 mg/mL stock.
• Instrument platform: Thermo Scientific Orbitrap Apex Tribrid mass spectrometer equipped with Direct Mass Technology (DMT) CDMS capability, ETD, and PTCR reagent ion sources. Heated electrospray ionization (HESI) produced positive analyte ions; built-in negative ion source generated ETD (fluoranthene) and PTCR (perfluoroperhydrophenanthrene) reagent ions. Quadrupole isolation and HCD fragmentation were used as appropriate.
• Acquisition strategy: Precursor envelope centered at m/z ~1002 (~29+ charge state) was isolated with a 4 Th window, ions were accumulated to 150% of the AIC target, reacted with ETD reagent for 1.25 ms, subjected to HCD at 25% normalized collision energy, and optionally exposed to PTCR (reported experiments used a 12 ms PTCR step; a 3 ms PTCR step was examined for charge-assignment effects). CDMS transients were recorded in the frequency domain and processed with STORIboard and Proteoform Studio.
• Data processing: Stringent ion filtering criteria were applied to reduce false positives (example thresholds: R2 ≥ 0.90, minimum duration and timing constraints, S/N threshold ~1, voting/binning parameters). Charge assignment compared a voting algorithm (high accuracy for separable envelopes) and a central-limit algorithm (used for unresolved distributions).

Main results and discussion

• Increased ion sampling and charge assignments: Across 1,600 spectra, datasets without PTCR yielded ~1.85 million ion signals with 111,949 confidently charge-assigned ions. With PTCR, ~2.82 million ion signals were recorded and 312,376 ions were charge-assigned — an ~2.8-fold increase in charge-assigned ions under identical acquisition counts.
• Redistribution to higher m/z and reduced congestion: PTCR shifted ions into a broader, higher m/z range, lowering local signal density in the low m/z region. This allowed longer injection windows under AIC control and increased the number of single-ion events sampled per acquisition without raising multi-ion overlap risk.
• Improved detection of high-mass fragments: High-mass, highly charged fragment ions that competed with abundant low-mass species in the congested low m/z region became detectable after PTCR. Comparative spectra showed substantial signal gain for high-mass fragments when PTCR was applied; fragment signal gain increased systematically with fragment mass, improving near-terminal fragment coverage driven by high-mass products.
• Sequence coverage gains: For bCA II proteoforms analyzed over 1,600 spectra, sequence coverage without PTCR ranged from 21.2% to 56.2%; with PTCR coverage increased to 39.0%–82.9%. The largest improvements localized near protein termini and were attributable to detection of high-mass fragments.
• Longer ion lifetimes: Charge-reduced ions exhibited lower kinetic energy and fewer collisional losses, translating to longer analyzer lifetimes and improved detection probability in CDMS transients.
• Enhanced charge assignment accuracy: Redistribution decreased interference from heterogeneous species and improved performance of charge-determination algorithms. As an example, an isolated precursor envelope containing two proteoforms centered at 29.05 kDa (88.63%) and 30.00 kDa (11.47%) was analyzed. The central-limit algorithm assigned only 15.55% of ions to the 29.05 kDa species without PTCR but 57.56% after a 3 ms PTCR step, demonstrating a marked improvement in robustness of the central-limit approach when spectra are decongested by PTCR.
• Filtering and quality control: Application of rigorous temporal, S/N and voting-based filters retained the majority of candidate ion signals while removing likely false positives; reported filtering removed a substantial fraction of raw events but preserved a higher absolute number of charge-assigned ions in the PTCR datasets.

Benefits and practical applications of the method

• Higher throughput: Increasing ions per spectrum reduces the number of acquisitions required to reach statistically robust ion counts for confident mass measurements, shortening overall analysis time for top-down and native MS experiments.
• Improved sensitivity for high-mass fragments: PTCR enables detection of fragments that would otherwise be suppressed in congested m/z regions, improving proteoform characterization and mapping of near-terminal sequence regions.
• Greater confidence in charge assignment: Especially for heterogeneous samples and cases where isotope resolution is poor, PTCR makes central-limit and other algorithms more reliable, reducing mass-assignment errors.
• Compatibility with existing workflows: PTCR is implemented as a brief gas-phase reaction step prior to Orbitrap injection and integrates with established fragmentation schemes (ETD, HCD, EThcD), making it straightforward to adopt on platforms that support reagent-ion chemistry.

Future trends and potential applications

• Optimization of PTCR conditions: Systematic tuning of reagent-ion types, reaction times, and AIC thresholds could further increase throughput and minimize side reactions or excessive neutralization for different analyte classes.
• Algorithmic co-development: Development of charge-assignment and filtering algorithms explicitly designed for PTCR-decongested CDMS data could further reduce false positives and exploit the larger ion counts per spectrum.
• Extension to larger assemblies: Applying PTCR-enabled CDMS to very large protein complexes, viral particles, or heterogeneous biotherapeutics could provide improved mass characterization where ensemble MS fails.
• Hybrid approaches: Combining PTCR with alternative ion-manipulation strategies (e.g., ion mobility or complementary ion/ion chemistries) may provide multi-dimensional separation that further boosts CDMS performance for complex samples.

Conclusion

Integrating proton transfer charge reduction with Orbitrap-based CDMS substantially mitigates spectral congestion by redistributing ions to higher m/z regions. This enables more ions per acquisition under AIC control, increases the number of confidently charge-assigned ions (~2.8-fold in the reported bCA II study), improves detection of high-mass fragments, and enhances sequence coverage in top-down proteomics. Importantly, PTCR markedly improves the performance of charge-assignment algorithms such as the central-limit method for heterogeneous envelopes. Overall, PTCR-augmented CDMS offers a practical route to higher-throughput, higher-sensitivity, and more reliable mass measurements for large and heterogeneous biomolecular systems.

Used instrumentation

• Thermo Scientific Orbitrap Apex Tribrid mass spectrometer with Direct Mass Technology (DMT) CDMS capability.
• Built-in negative reagent-ion source for ETD (fluoranthene) and PTCR (perfluoroperhydrophenanthrene).
• Heated electrospray ionization (HESI) for analyte generation; quadrupole isolation; HCD and ETD fragmentation modalities; data processed with STORIboard and Proteoform Studio.

Reference

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