Development of timsMRMS: A Gated-TIMS FT-ICR MS instrument to decipher isomeric content of complex organic mixtures

Significance of the topic

Bridging high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS / MRMS) with trapped ion mobility spectrometry (TIMS) addresses a longstanding gap in comprehensive structural analysis of complex organic mixtures. Combining TIMS separation with ultra-high-resolution FT-ICR detection enables separation of isomeric and conformational variants before high-accuracy mass measurement and electron-based dissociation (ExD) MS/MS, improving confidence in compositional assignment for environmental samples, bio-oils, proteomics, and imaging mass spectrometry.

Objectives and overview of the study

The work presents the development and characterization of a new integrated instrument (timsMRMS or gTIMS-MRMS) that combines gated-TIMS (gTIMS) with a ParaCell FT-ICR mass analyzer and advanced front-end hardware. Key objectives were to:

• Integrate modern dual-accumulation TIMS hardware and gated operation with an FT-ICR MS platform optimized for high ion flux and wide m/z transmission.
• Maintain ultra-low-energy ion transmission to preserve fragile structures and enable downstream ExD fragmentation.
• Enable high-spatial-resolution MALDI imaging with adaptive focusing and rapid laser rastering.
• Demonstrate fidelity of mobility separations, high dynamic range detection, and operational modes for complex mixture analysis and protein conformer-selective MS/MS.

Used Instrumentation

• timsMRMS prototype combining a gated TIMS module (D7 TIMS cartridge) with an 18 T FT-ICR ParaCell analyzer.
• Dual ion sources: electrospray ionization (ESI) and MALDI, software-switchable.
• SmartBeam 3D MALDI laser with galvo-controlled beam scanning (5 µm fixed focus capability) and a microgrid XYZ in-vacuum sample stage with micron-level encoders and an independent height-measurement laser for adaptive focus.
• Fast-switching quadrupole re-purposed as an ion gate for gated-TIMS (gTIMS) operation enabling simultaneous mobility and m/z selection.
• Hexapole-style collision cell and a 21-lens stacked lens assembly to support large ion capacity, wide m/z transmission on single voltage profiles, low-energy storage, and high ejection performance.

Methodology and experimental approach

The instrument architecture was designed to preserve ion integrity and maximize duty cycle and sensitivity. Key methodological elements included:

• Dual-accumulation TIMS funnel architecture to run accumulation and analysis in parallel, approaching nearly 100% duty cycle and enabling continuous throughput of TIMS-separated ions to the collision cell and ICR.
• Gated TIMS modes: (1) sweeping gate—iteratively translating a binary gate over a repeating TIMS ramp, and (2) sweeping ramp—keeping a static gate while shifting the TIMS ramp between scans. Both approaches were validated for mobility fidelity.
• Optimization of TIMS internal voltages (T1–T4) and gas flow management across two pumping stages to reduce mobility-dependent activation and provide a uniform low-energy transfer profile.
• Implementation of hexapole collision cell multi-filling: accumulating mobility- and m/z-selected ions across multiple TIMS cycles to the capacity of the collision cell before ExD fragmentation to maximize linear signal-to-noise and enable transient summation strategies for S/N improvement.
• Evaluation of ion storage impact on ExD fragmentation: comparing immediate transfer (ICT = 0 s) versus additional multi-cycle trapping (ICT = 2 s) prior to ExD MS/MS.
• MALDI imaging using SmartBeam 3D laser with galvo scanning and microgrid stage to achieve tuneable pixel sizes down to 10 µm spatial resolution with dynamic focus correction from a height-measurement laser.

Main results and discussion

• Mobility fidelity and precision: Both gated modes (sweeping gate and sweeping ramp) demonstrated excellent mobility linearity and reproducibility with calibration showing mobility precision of R2 ≈ 0.9999 for tested species.
• High dynamic range and ion capacity: The front-end and ParaCell chain enabled accumulation of very large ion ensembles—up to ~15 million ions per scan reported—and an intra-scan dynamic range example of about 5.1 orders of magnitude, extended to ~5.4 orders with hexapole collision cell and stacked lens improvements.
• Low-energy transmission and stable storage: The hexapole collision cell provided low-energy, stable ion storage for tens of seconds without heating or loss, enabling multiple fillings and consistent ExD fragmentation behavior. Storage time experiments comparing ICT = 0 s and ICT = 2 s showed no measurable differences in ExD fragmentation profiles, supporting multi-filling accumulation strategies.
• Parallel accumulation benefits: Running accumulation and analysis concurrently allowed nearly continuous operation, enabling efficient build-up of signal by repeating TIMS fills into the collision cell and leveraging transient summation across ICR transients for root(n) improvements in S/N.
• MALDI imaging performance: Integration of SmartBeam 3D, galvo rastering, and microgrid stage produced consistent ablation spot quality and enabled routine 10 µm spatial resolution imaging on tissue with high resolving power (e.g., ~800k at m/z 400 reported for selected experiments).
• Applicability to complex matrices: Demonstrations with environmental extracts, Suwannee River fulvic acid (SRFA), and bio-oil type samples showed that mobility width information can help infer isomeric content within complex organic mixtures at lower charge states per scan, paving the way for collision cross section (CCS)-informed composition analysis.

Benefits and practical applications

• Deciphering isomeric and conformational complexity: Pre-separation by TIMS prior to FT-ICR allows resolving species that would be indistinguishable by m/z alone, improving structural assignments in environmental, petroleomic, and biochemical samples.
• High-resolution MS/MS of mobility-selected ions: The system supports ExD fragmentation of mobility-separated ions, enabling conformer-selective structural probing (e.g., protein unfolding and conformer-specific MS/MS).
• High dynamic range analyses: Ability to accumulate across multiple TIMS cycles and store large ion populations supports detection of low-abundance components within complex mixtures without compromising mass accuracy or resolution.
• Flexible imaging workflows: Software-switchable ESI/MALDI sources and adaptive MALDI focusing enable high-resolution spatially-resolved molecular imaging for tissue and materials studies.
• Improved throughput: Parallel TIMS accumulation/analysis and efficient gating strategies enhance throughput for large sample sets while maintaining analytical performance.

Future trends and potential uses

• Integration of CCS-informed databases and algorithms to combine mobility-derived CCS values with ultra-high mass accuracy for more confident identification of isomers and structural motifs in complex mixtures.
• Expanded ExD workflows: Further optimization of ExD fragmentation directly following mobility separation for comprehensive gas-phase structural characterization of proteins, lipids, and complex organics.
• Automation of multi-cycle accumulation strategies and advanced transient summation to push detection limits while maintaining high resolution for routine analyses.
• MALDI imaging advances: Pushing spatial resolution below 10 µm combined with mobility separation could enable sub-cellular molecular imaging with high confidence in species assignments.
• Application expansion: Broader adoption in environmental chemistry, renewable energy feedstock characterization (bio-oils), complex natural product mixtures, and spatial proteomics.

Conclusion

The timsMRMS (gTIMS-MRMS) platform demonstrates that combining gated-TIMS separation with an optimized FT-ICR MS front-end and ParaCell analyzer yields a powerful system for resolving isomeric and conformational complexity in challenging samples. Key strengths are ultra-low-energy transmission, high ion capacity with stable storage, high mobility precision, and flexible imaging capabilities. These improvements enable mobility-selected ExD MS/MS, superior dynamic range, and practical workflows for complex mixture analysis and high-resolution MALDI imaging. Continued integration with CCS databases, automated accumulation strategies, and expanded ExD methods will further enhance the platform’s utility across analytical chemistry applications.

References

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