Novel MALDI-TOF MS Workflow for Ultrahigh-Throughput Screening of Different Analytes at Each Position on a Plate

Significance of the Topic

Ultrahigh-throughput screening of small molecules is critical in drug discovery and reaction optimization. Extending MALDI-TOF MS workflows to handle diverse analytes at each spot on a plate enables rapid evaluation of compound libraries and chemical transformations without labels or chromatographic separation.

Objectives and Overview of the Study

The study presents the novel Synthesis Screening workflow in MALDI PharmaPulse software applied to a custom library of 41 small molecule pharmaceuticals. It aims to demonstrate automated, high-density plate screening in 384 format, monitoring up to 100 analytes per position and achieving minimal failed spots.

Methodology

The workflow involves spotting each compound eight times on a 384-well MALDI plate, overlaying with a solution of alpha-cyano-4-hydroxycinnamic acid matrix, and performing automated analysis.

• The plate format allows 384 or higher density layouts.
• Each spot yields 2000 laser shots per spectrum.
• Custom analyte lists enable monitoring of different compounds at each position.

Used Instrumentation

• rapifleX MALDI-TOF/TOF system with 10 kHz scanning beam laser
• MALDI PharmaPulse 2.2 software with Synthesis Screening module
• 384-well stainless steel MALDI plates

Main Results and Discussion

The automated runs across three plates averaged fewer than one failed position per run. One run had zero failures and two runs each had one. The software successfully detected all 41 compounds in 328 positions.

• Five compounds were monitored as sodium adducts while the remainder were detected as protonated species.
• Representative spectra (mass range 155–760 Da) showed high signal quality and reproducibility, exemplified by azithromycin at m/z 749.516 and other standards.
• Real-time plate view links each spot to its extracted spectrum and signal parameters.
• Relative quantitation options support a single internal standard for the entire plate or unique standards per position.

Benefits and Practical Applications

The workflow offers:

• Label-free, high-throughput analysis of diverse compound libraries
• Customizable analyte lists enabling different targets at each spot
• Integration of screening and QC in one automated run
• Compatibility with various plate densities and internal standard schemes

Future Trends and Potential Applications

Advancements may include integration with AI-driven data analysis, expansion to higher density formats, coupling with microfluidic platforms, and broader application to multiplexed biochemical or cell-based assays. Further software enhancements could support real-time decision making in synthetic chemistry and accelerated lead optimization.

Conclusion

The novel Synthesis Screening module in MALDI PharmaPulse software enables reliable, ultrahigh-throughput screening of different small molecule analytes at each plate position. It demonstrates minimal failure rates, high data quality, and flexible quantitation, making it a valuable tool in pharmaceutical library screening and reaction monitoring.

References

[1] Haslam C Hellicar J Dunn A Fuetterer A Hardy N Marshall P Paape R Pemberton M Resemann A Leveridge MJ J Biomol Screen 21 176–186 2016
[2] Heap RE Hope AG Pearson LA Reyskens KMSE McElroy SP Hastie CJ Porter DW Arthur JSC Gray DW Trost M SLAS Disc 10 1193–1202 2017
[3] Lin S Dikler S Blincoe WD Ferguson RD Sheridan RP Peng Z Conway DV Zawatzky K Wang H Cernak T Davies IW DiRocco DA Sheng H Welch CJ Dreher SD Science 361 eaar6236 2018