Label-free compound profiling in K562 leukemia cells by MALDI-TOF mass spectrometry using heme B as response marker

Importance of the Topic

This study highlights the application of label-free MALDI-TOF mass spectrometry for rapid, high-throughput screening of kinase inhibitors directly in whole cells. By avoiding fluorogenic labels and antibodies, this approach overcomes common challenges such as autofluorescence and reduces assay complexity, offering a versatile platform for pharmacodynamic profiling in drug discovery.

Study Objectives and Overview

The primary aim was to develop and validate a cell-based assay for quantifying the effect of BCR-ABL inhibitors on K562 leukemia cells. Heme B was selected as a phenotypic marker of erythroid redifferentiation, allowing establishment of concentration-response relationships for imatinib, dasatinib and a negative control (chloroquine).

Methodology and Instrumentation

The workflow combined automated liquid handling with MALDI-TOF MS analysis:

• Cell culture: K562 cells in 96-well plates at 1×10^5 cells/mL, treated for 48 h with compounds or DMSO control.
• Automation: Compound dispensing and cell transfer to a 384-spot steel MALDI target via CyBio FeliX robot.
• Matrix deposition: 20 mg/mL DHB in 1:1 ACN/water with 0.5% TFA using an HTX TM-sprayer.
• Mass analysis: Bruker rapifleX MALDI-TOF MS (reflector, positive mode, m/z 500–1000, 2 kHz laser) and solariX XR 7 T MRMS for signal validation.
• Data processing: flexAnalysis for recalibration, intensity extraction, and GraphPad Prism for dose-response fitting.

Main Results and Discussion

The assay delivered a clear, concentration-dependent increase of the heme B signal (m/z 616.2) upon treatment with BCR-ABL inhibitors, with no interference from unrelated compounds. Ultra-high-resolution MRMS confirmed the specificity of the peak as heme B. Calculated pIC50 values were 7.1 ± 0.1 for imatinib and 9.8 ± 0.1 for dasatinib, correlating well with published data.

Benefits and Practical Applications

• Label-free detection reduces assay cost and eliminates secondary reagents.
• Automated sample handling improves throughput, reproducibility and scalability.
• Direct analysis of whole cells bypasses cleanup steps, streamlining workflow.
• Capability to screen pharmacodynamics at lipid and metabolite level enables multiplexed readouts.

Future Trends and Possibilities

Emerging directions include expansion to diverse cell types and targets, integration with high-content MALDI imaging, computational lipidomics for multiplex biomarker panels, and incorporation of AI-driven data analysis to accelerate hit identification.

Conclusion

The rapifleX MALDI-TOF MS platform, when combined with automated liquid handling, provides a robust, rapid and reproducible cell-based assay for drug screening. Heme B serves as a reliable phenotypic marker, and the approach aligns closely with reference cell-based methods while offering significant operational advantages.

References

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