Developing a declustering ion guide and Cyclic IMS-enabled Wideband Enhancement for native top-down studies

Significance of the topic

Native top-down mass spectrometry (MS) enables the characterization of intact proteoforms and noncovalent assemblies in a biologically relevant state, preserving ligand and lipid interactions. However, incomplete desolvation and adduction from salts, buffers and detergents reduce spectral resolution and sensitivity, limiting confident sequence assignment and analysis of low-abundance proteoforms and large assemblies. Improvements in front-end ion declustering and enhanced TOF sampling can therefore materially extend the practical scope of native top-down workflows, especially for membrane proteins and heterogeneous complexes.

Objectives and overview of the study

This work reports the development and evaluation of two complementary hardware/software strategies to improve native top-down analysis on a Cyclic IMS platform: (1) a Dynamic Field Declustering (DFD) ion guide positioned pre-IMS to promote effective desolvation and declustering of native ions, and (2) a Wideband Enhancement (WBE) mode that synchronizes ion release with TOF pulsing to increase duty cycle and sensitivity across a broad m/z range. Experiments used NISTmAb as an optimization standard and demonstrated applications to streptavidin top-down pseudo-MS3 and release/characterization of bacteriorhodopsin (bR) from detergent micelles to identify bound lipids.

Methodology

• Samples: NISTmAb (reference antibody), streptavidin, and bacteriorhodopsin from Halobacterium salinarum. Native sprays used 200 mM ammonium acetate. bR was prepared in 40 mM octyl-β-D-glucopyranoside (OG) detergent and buffer-exchanged into 200 mM ammonium acetate.
• Ionization: Nanoelectrospray (nESI) from 2 µm ID borosilicate capillaries.
• DFD optimization: NISTmAb spectra were recorded at increasing DFD amplitudes (0–280 Vpp) at a fixed frequency (60 kHz) to assess declustering and apparent glycoform resolution.
• Top-down workflow: Streptavidin tetramer was declustered with DFD (example 175 Vpp, 60 kHz), partial monomer dissociation allowed quadrupole selection of a monomer charge state followed by CID (pseudo-MS3) and WBE-enabled TOF acquisition to maximize sampling efficiency and sequence coverage.
• bR analysis: DFD parameters were tuned to remove detergent micelle adducts and reveal monomeric and lipid-bound species with isotopic resolution; deconvolution used top-down processing in UNIFI.

Instrumentation

• Waters Cyclic IMS P20 Mass Spectrometer operated in positive nESI mode.
• Dynamic Field Declustering (DFD) ion guide installed in the pre-IMS region to apply oscillatory fields for ion declustering and partial activation.
• Wideband Enhancement (WBE) mode implemented to synchronize ion packet release from IMS with TOF pusher timing.
• Data acquisition and processing: MassLynx v4.2 and waters_connect UNIFI top-down workflow.

Main results and discussion

• DFD improves declustering and apparent spectral resolution: Increasing DFD amplitude (0 to 280 Vpp at 60 kHz) progressively reduced solvent/salt adducting and sharpened NISTmAb glycoform peaks, demonstrating robust desolvation with minimal tuning.
• Streptavidin pseudo-MS3 top-down: Using DFD (example 175 Vpp, 60 kHz) produced improved resolution of the tetramer and promoted partial ejection of a 7+ monomer. The monomer was quadrupole-selected, subjected to CID, and analyzed with WBE-enabled TOF acquisition, yielding high-quality fragment spectra and 78.7% sequence coverage for the monomer.
• WBE increases effective TOF duty cycle and sensitivity: By releasing mobility-separated ion packets in synchrony with the TOF push, WBE markedly improved sampling efficiency for cyclic IMS experiments across a broad m/z range, translating into greater tandem MS sensitivity and faster acquisition of high-quality top-down spectra.
• Membrane protein declustering and lipid detection: DFD facilitated removal of OG detergent micelle adducts from bacteriorhodopsin, revealing isotopically resolved monomeric bR (e.g., 9+ charge state) and enabling deconvolved detection of lipid-bound forms. Identified archaeal lipids included PGP-Me and S-TGA-1 consistent with known purple membrane composition.
• Instrumental interplay: The combination of DFD (to decluster and release intact subunits/ligands) and WBE (to maximize ion sampling during TOF detection) enables pseudo-MS3 top-down experiments directly from native complexes, improving sequence-level information without lengthy method development.

Benefits and practical applications

• Improved native MS throughput: DFD reduces the need for extensive buffer exchange or harsh activation to remove adducts, streamlining sample handling for native analyses.
• Enhanced sensitivity for top-down: WBE raises effective TOF duty cycle, particularly beneficial for low-abundance proteoforms and broad-mass-range separations produced by IMS.
• Membrane protein analysis: DFD enables release of membrane proteins from detergent micelles while preserving specific lipid interactions, facilitating direct detection and assignment of bound lipids and proteoform heterogeneity.
• Pseudo-MS3 workflows: The combined approach allows quadrupole isolation of subunits followed by fragmentation to obtain sequence information from intact assemblies, useful for proteoform mapping and complex characterization in structural proteomics, biopharma QA/QC and lipidomics.

Future trends and possibilities for use

• Complex-level top-down: Extending DFD/WBE strategies to preserve and interrogate higher-order oligomers (e.g., trimers, tetramers) with optimized sample prep to access intact-complex top-down sequencing.
• Broader membrane proteome coverage: Integrating refined detergent/lipid exchange protocols and DFD tuning to analyze a wider range of membrane protein classes with native lipid complements.
• Advanced activation schemes: Combining DFD with alternative activation methods (ECD/ETD, UVPD, electron-based dissociation) to improve fragmentation coverage for labile regions while retaining noncovalent interactions.
• Software and duty-cycle optimization: Further development of WBE control algorithms and synchronization strategies to maximize transmission across even larger m/z windows and faster IMS cycles.
• High-throughput native top-down pipelines: Automation of DFD parameters, standardized WBE templates and integration into data-analysis workflows for routine proteoform-centric quality control in biopharma and structural biology labs.

Conclusions

The Dynamic Field Declustering guide and Wideband Enhancement mode together provide a practical, complementary enhancement to Cyclic IMS-enabled native top-down workflows. DFD efficiently removes solvent, salt and detergent adducts and can liberate membrane proteins from micelles while preserving bound lipids; WBE increases TOF duty cycle and sensitivity by synchronizing ion packet release with the pusher. In combination these technologies enable pseudo-MS3 top-down sequencing of native assemblies with substantially improved sequence coverage and throughput, broadening the applicability of native MS to challenging proteoforms and membrane proteins. Ongoing work will focus on sample preparation and method refinements to access intact multimers and further expand native top-down capability.

Reference

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