Label-free drug uptake analysis of whole cells by MALDI-TOF mass spectrometry using the transport protein OATP2B1 as an example

Importance of the Topic

Transport proteins such as OATP2B1 play a central role in drug absorption, distribution, and elimination and are implicated in drug-drug interactions and various human diseases. Conventional assays for transporter function rely on radiolabeled or fluorescent substrates, which present challenges in waste handling, safety, potential photonic artefacts, and false positives or negatives. A label-free, high-throughput method is therefore desirable to screen drug uptake and inhibition directly in whole cells, improving throughput, reproducibility, and safety.

Objectives and Study Overview

This study aimed to develop and validate a MALDI-TOF mass spectrometry assay for quantifying cellular uptake of estrone-3-sulfate (E3S) mediated by OATP2B1 and to identify inhibitors among a library of 294 marketed and known transporter-related compounds. The goals were to demonstrate rapid screening capabilities, determine precise IC50 values, and benchmark results against established fluorescence- and radiolabel-based assays.

Methodology and Instrumentation

HEK293 cells overexpressing OATP2B1 or carrying a vector control were cultured and seeded into 96-well plates. After inducing transporter expression with sodium butyrate and serum starvation, cells were incubated with test compounds and E3S. Following brief substrate uptake, cells were washed, snap-frozen, and stored at –80°C. For analysis, wells were resuspended in water containing a deuterated E3S internal standard and matrix solution (Ph-CCA-NH2 in acetonitrile) was applied via automated dried-droplet deposition. MALDI-TOF measurements were performed on a rapifleX spectrometer using MALDI Pharma Pulse software in negative reflector mode (300–900 m/z). pIC50 values were calculated with GraphPad Prism, using erlotinib as positive and DMSO as negative controls.

Instrumentation

• rapifleX MALDI-TOF mass spectrometer
• MALDI Pharma Pulse (MPP) software
• CyBio FeliX liquid handling and dried droplet application platform
• GraphPad Prism software for pIC50 determination

Main Results and Discussion

A single-point screen of 294 compounds identified 67 initial hits (≥50% inhibition), of which 14 exhibited potent inhibition (pIC50 ≥ 6). The assay showed intra-assay precision with mean CV below 10% and standard deviation of 0.3, underscoring robustness and reproducibility. Comparison with fluorescence-based and radiolabeled assays revealed an 83% overlap in inhibitor identification, confirming the method’s reliability. The optimized Ph-CCA-NH2 matrix provided clear analyte peaks without interference, outperforming traditional DHAP matrix in signal clarity.

Benefits and Practical Applications

• Label-free detection eliminates safety and waste issues associated with radiolabels.
• Avoids photonic artefacts common to fluorescence assays.
• Automated sample preparation and rapid measurement (~5 minutes per 384-well plate) support high throughput screening.
• Quantitative pIC50 determination directly in intact cells enables accurate assessment of drug-transporter interactions.

Future Trends and Possibilities

Advancements in MALDI MS automation and data integration are expected to expand cell-based screening for diverse transporters and biochemical pathways. Coupling MALDI MS with high-content imaging or single-cell analysis may provide multidimensional insights into cellular responses. Integration with artificial intelligence for spectral interpretation and predictive modeling could further accelerate transporter drug interaction studies and personalized medicine applications.

Conclusion

This work establishes a robust MALDI-TOF MS assay for label-free analysis of OATP2B1-mediated drug uptake in whole cells. The approach compares favorably to established methods, offers rapid high-throughput capability, and accurately determines inhibitor potency. It represents a significant step toward safer, faster, and more reliable screening of transporter-based drug interactions.

Reference

1. Unger MS et al. Direct Automated MALDI Mass Spectrometry Analysis of Cellular Transporter Function: Inhibition of OATP2B1 Uptake by 294 Drugs. Anal Chem. 2020;92:11851–11859.
2. Beeman K et al. In Situ MALDI-Based High-Throughput Screening: Case Study with c-MET. SLAS Discov. 2017;22:1203–1210.
3. Simon RP et al. MALDI-TOF MS High-Throughput Screening for cGAS Inhibitors. SLAS Discov. 2019.
4. Weigt D et al. Mechanistic MALDI-TOF Cell-Based Assay for Fatty Acid Synthase Inhibitors. Cell Chem Biol. 2019;26:1322–1331.
5. Unger MS et al. Clinically Relevant OATP2B1 Inhibitors in Marketed Drug Space. Mol Pharm. 2020.
6. Karlgren M et al. Classification of OATP Inhibitors: Influence of Protein Expression on DDIs. J Med Chem. 2012;55:4740–4763.