High-throughput experimentation reaction monitoring and analysis of chemistry synthesis products with ultrahigh-throughput 4D timsTOF fleX MALDI-2 technology

Significance of the Topic

High-throughput reaction monitoring reduces turnaround time and resource consumption in drug discovery by providing near real-time feedback on chemical synthesis outcomes. MALDI-2 coupled with trapped ion mobility spectrometry and ultrahigh-resolution time-of-flight mass spectrometry enables rapid, sensitive, and multidimensional analysis of small molecules with minimal sample consumption and high automation potential.

Study Objectives and Overview

This work demonstrates the application of 4D timsTOF fleX MALDI-2 technology for high-throughput experimentation (HTE) reaction monitoring. It focuses on:

• Confident identification of synthesis products using four physical attributes: collisional cross-section (CCS), accurate mass, isotopic pattern, and MS/MS fragmentation.
• Assessment of throughput, sensitivity, and reproducibility for panels of drug reference compounds and reaction mixtures.
• Evaluation of CCS predictive models for isomer discrimination and compound confirmation.

Methodology and Instrumentation

Microliter aliquots of drug standards and 95 chemical synthesis reactions were prepared in DMSO, diluted, spotted on MALDI plates, and overlaid with HCCA or 2,5-DHAP matrices. Data acquisition employed a Bruker timsTOF fleX with:

• Dual ESI/MALDI ion source with MALDI-2 post-ionization.
• Smartbeam 3D laser at 10 kHz for ultrahigh throughput.
• Trapped ion mobility spectrometry (TIMS) with 1/K0 gradient and 150 ms cycle time.
• Ultrahigh-resolution TOF mass analyzer for sub-ppm accuracy and true isotopic pattern fidelity.

Data processing was conducted in DataAnalysis 5.3 for peak picking, isotope matching, ion mobilogram extraction, and quantitative analysis via custom scripts.

Main Results and Discussion

The 4D MALDI-2 method achieved:

• Acquisition speed of ~1 s per sample in MALDI-MS mode and an additional 100–300 ms per TIMS frame.
• Mass accuracy ≤1 ppm and mSigma values indicating excellent isotope fidelity.
• Successful detection of 93% of expected products in a 95-reaction panel, with TIMS resolving isobaric interferences and enabling unambiguous identification.
• Highly reproducible CCS measurements (RSD ~0.07%) matching predicted values within 0.1–5.3%.
• Linear quantitation over two orders of magnitude for selected drug mixtures, using verapamil as an internal standard.
• Enhanced ionization of challenging analytes, such as zafirlukast and estradiol, with >5-fold signal increase using MALDI-2.

Benefits and Practical Applications

• Rapid, automated screening of chemical libraries with minimal sample volumes (picomoles per spot).
• Orthogonal TIMS separation enhances confidence in compound identity by removing matrix and isobaric interferences.
• 4D data (CCS, mass, isotope pattern, MS/MS) allows robust confirmation without prior reference spectra.
• MALDI-2 expands analyte coverage to molecules previously inaccessible by conventional MALDI.

Used Instrumentation

• Bruker timsTOF fleX MALDI-2 platform
• Smartbeam 3D laser (10 kHz)
• Trapped ion mobility spectrometer (TIMS)
• Ultrahigh-resolution TOF mass analyzer
• timsControl 2.0 and DataAnalysis 5.3 software

Future Trends and Applications

Emerging trends include the integration of CCS predictive models for rapid isomer discrimination, expansion of MALDI-2 applications to lipidomics and metabolomics, and incorporation into spatial omics workflows. Further automation and miniaturization will advance real-time feedback in HTE, reducing resource consumption and accelerating lead discovery and optimization.

Conclusion

The 4D timsTOF fleX MALDI-2 technology provides an ultrafast, sensitive, and multidimensional platform for high-throughput reaction monitoring in drug discovery. Its combination of high throughput, low sample consumption, orthogonal separations, and enhanced ionization addresses key bottlenecks in synthesis screening and offers a scalable solution for modern analytical challenges.

References

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