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Label-free cell assays to determine compound uptake or drug action using MALDI-TOF mass spectrometry


Cell-based assays for compound screening and profiling are fundamentally important in life sciences, chemical biology and pharmaceutical research. Most cell assays measure the amount of a single reporter molecule or cellular endpoint, and require the use of fluorescence or other labeled materials. Consequently, there is high demand for label-free technologies that enable multiple biomolecules or endpoints to be measured simultaneously. Here, we describe how to develop, optimize and validate MALDI-TOF mass spectrometry (MS) cell assays that can be used to measure cellular uptake of transporter substrates, to monitor cellular drug target engagement or to discover cellular drug-response markers. In uptake assays, intracellular accumulation of a transporter substrate and its inhibition by test compounds is measured. In drug response assays, changes to multiple cellular metabolites or to abundant posttranslational protein modifications are monitored as reporters of drug activity. We detail a ten-part optimization protocol with every part taking 1–2 d that leads to a final 2 d optimized procedure, which includes cell treatment, transfer, MALDI MS-specific sample preparation, quantification using stable-isotope-labeled standards, MALDI-TOF MS data acquisition, data processing and analysis. Key considerations for validation and automation of MALDI-TOF MS cell assays are outlined. Overall, label-free MS cell-based assays offer speed, sensitivity, accuracy and versatility in drug research.

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Fig. 1: Applications of cell-based MALDI-TOF MS assays.
Fig. 2: Schematic overview of the general label-free MALDI MS cell assay workflow.
Fig. 3: Development of a cell-based MALDI-TOF MS assay—possible output of the protocol.
Fig. 4: Possible output of untargeted MALDI MS cell-based assays.

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Data availability

The raw data that support the anticipated results are available at figshare: Additional data are available from the corresponding author upon reasonable request.

Code availability

The in-house R scripts are publicly available on the CeMOS GitHub at


  1. Horvath, P. et al. Screening out irrelevant cell-based models of disease. Nat. Rev. Drug Discov. 15, 751–769 (2016).

    Article  CAS  PubMed  Google Scholar 

  2. Thakare, R., Chhonker, Y. S., Gautam, N., Alamoudi, J. A. & Alnouti, Y. Quantitative analysis of endogenous compounds. J. Pharm. Biomed. Anal. 128, 426–437 (2016).

    Article  CAS  PubMed  Google Scholar 

  3. Gordon, L. J. et al. Direct measurement of intracellular compound concentration by rapidfire mass spectrometry offers insights into cell permeability. J. Biomol. Screen. 21, 156–164 (2016).

    Article  CAS  PubMed  Google Scholar 

  4. Karas, H., Bachmann, D. & Hillenkamp, F. Influence of the wavelength in high-irradiance ultraviolet laser desorption mass spectrometry of organic molecules. Anal. Chem. 57, 2935–2939 (1985).

    Article  CAS  Google Scholar 

  5. Stoeckli, M., Chaurand, P., Hallahan, D. E. & Caprioli, R. M. Imaging mass spectrometry: a new technology for the analysis of protein expression in mammalian tissues. Nat. Med. 7, 493–496 (2001).

    Article  CAS  PubMed  Google Scholar 

  6. Schulz, S., Becker, M., Groseclose, M. R., Schadt, S. & Hopf, C. Advanced MALDI mass spectrometry imaging in pharmaceutical research and drug development. Curr. Opin. Biotechnol. 55, 51–59 (2019).

    Article  CAS  PubMed  Google Scholar 

  7. Ly, A. et al. High-mass-resolution MALDI mass spectrometry imaging of metabolites from formalin-fixed paraffin-embedded tissue. Nat. Protoc. 11, 1428–1443 (2016).

    Article  PubMed  Google Scholar 

  8. Casadonte, R. & Caprioli, R. M. Proteomic analysis of formalin-fixed paraffin-embedded tissue by MALDI imaging mass spectrometry. Nat. Protoc. 6, 1695–1709 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. RamalloGuevara, C., Paulssen, D., Popova, A. A., Hopf, C. & Levkin, P. A. Fast nanoliter-scale cell assays using droplet microarray-mass spectrometry imaging. Adv. Biol. (Weinh.) 5, e2000279 (2021).

    Article  Google Scholar 

  10. Haslam, C. et al. The evolution of MALDI-TOF mass spectrometry toward ultra-high-throughput screening: 1536-well format and beyond. J. Biomol. Screen. 21, 176–186 (2016).

    Article  CAS  PubMed  Google Scholar 

  11. Simon, R. P. et al. MALDI-TOF mass spectrometry-based high-throughput screening for inhibitors of the cytosolic DNA sensor cGAS. SLAS Discov. (2019).

  12. De Cesare, V. et al. High-throughput matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry-based deubiquitylating enzyme assay for drug discovery. Nat. Protoc. 15, 4034–4057 (2020).

    Article  PubMed  Google Scholar 

  13. Machálková, M., Schejbal, J., Glatz, Z. & Preisler, J. A label-free MALDI TOF MS-based method for studying the kinetics and inhibitor screening of the Alzheimer’s disease drug target β-secretase. Anal. Bioanal. Chem. 410, 7441–7448 (2018).

    Article  PubMed  Google Scholar 

  14. Beeman, K. et al. Integration of an in situ MALDI-based high-throughput screening process: a case study with receptor tyrosine kinase c-MET. SLAS Discov. 22, 1203–1210 (2017).

    Article  CAS  PubMed  Google Scholar 

  15. Simon, R. P. et al. MALDI-TOF-based affinity selection mass spectrometry for automated screening of protein-ligand interactions at high throughput. SLAS Discov. 26, 44–57 (2021).

    Article  CAS  PubMed  Google Scholar 

  16. Chandler, J., Haslam, C., Hardy, N., Leveridge, M. & Marshall, P. A systematic investigation of the best buffers for use in screening by MALDI-mass spectrometry. SLAS Discov. 22, 1262–1269 (2017).

    Article  CAS  PubMed  Google Scholar 

  17. Winter, M. et al. Chemical derivatization enables MALDI-TOF-based high-throughput screening for microbial trimethylamine (TMA)-lyase inhibitors. SLAS Discov. 24, 766–777 (2019).

    Article  CAS  PubMed  Google Scholar 

  18. Dong, H. et al. Rapid detection of apoptosis in mammalian cells by using intact cell MALDI mass spectrometry. Analyst 136, 5181–5189 (2011).

    Article  CAS  PubMed  Google Scholar 

  19. Zhang, X., Scalf, M., Berggren, T. W., Westphall, M. S. & Smith, L. M. Identification of mammalian cell lines using MALDI-TOF and LC-ESI-MS/MS mass spectrometry. J. Am. Soc. Mass Spectrom. 17, 490–499 (2006).

    Article  CAS  PubMed  Google Scholar 

  20. Munteanu, B., von Reitzenstein, C., Hansch, G. M., Meyer, B. & Hopf, C. Sensitive, robust and automated protein analysis of cell differentiation and of primary human blood cells by intact cell MALDI mass spectrometry biotyping. Anal. Bioanal. Chem. 404, 2277–2286 (2012).

    Article  CAS  PubMed  Google Scholar 

  21. Munteanu, B. et al. Label-free in situ monitoring of histone deacetylase drug target engagement by matrix-assisted laser desorption ionization-mass spectrometry biotyping and imaging. Anal. Chem. 86, 4642–4647 (2014).

    Article  CAS  PubMed  Google Scholar 

  22. Szaruga, M. et al. Alzheimer’s-causing mutations shift Aβ length by destabilizing γ-secretase-Aβn interactions. Cell 170, 443–456.e414 (2017).

    Article  CAS  PubMed  Google Scholar 

  23. Weigt, D., Sammour, D. A., Ulrich, T., Munteanu, B. & Hopf, C. Automated analysis of lipid drug-response markers by combined fast and high-resolution whole cell MALDI mass spectrometry biotyping. Sci. Rep. 8, 11260 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Weigt, D. et al. Mechanistic MALDI-TOF cell-based assay for the discovery of potent and specific fatty acid synthase inhibitors. Cell Chem. Biol. 26, 1322–1331.e1324 (2019).

    Article  CAS  PubMed  Google Scholar 

  25. Unger, M. S. et al. Direct automated MALDI mass spectrometry analysis of cellular transporter function: inhibition of OATP2B1 uptake by 294 drugs. Anal. Chem. 92, 11851–11859 (2020).

    Article  CAS  PubMed  Google Scholar 

  26. Galey, M. M. et al. Detection of ovarian cancer using samples sourced from the vaginal microenvironment. J. Proteome Res. 19, 503–510 (2020).

    Article  CAS  PubMed  Google Scholar 

  27. Winter, M. et al. Establishing MALDI-TOF as versatile drug discovery readout to dissect the PTP1B enzymatic reaction. SLAS Discov. 23, 561–573 (2018).

    Article  CAS  PubMed  Google Scholar 

  28. Zhou, Q., Fülöp, A. & Hopf, C. Recent developments of novel matrices and on-tissue chemical derivatization reagents for MALDI-MSI. Anal. Bioanal. Chem. (2020).

  29. Truong, K. & Ikura, M. The use of FRET imaging microscopy to detect protein-protein interactions and protein conformational changes in vivo. Curr. Opin. Struct. Biol. 11, 573–578 (2001).

    Article  CAS  PubMed  Google Scholar 

  30. Xu, Z. et al. Development of high-throughput TR-FRET and AlphaScreen assays for identification of potent inhibitors of PDK1. J. Biomol. Screen. 14, 1257–1262 (2009).

    Article  CAS  PubMed  Google Scholar 

  31. Ergin, E., Dogan, A., Parmaksiz, M., Elçin, A. E. & Elçin, Y. M. Time-Resolved Fluorescence Resonance Energy Transfer [TR-FRET] Assays for Biochemical Processes. Curr Pharm Biotechnol 17, 1222–1230 (2016).

    Article  CAS  PubMed  Google Scholar 

  32. Xu, Y., Piston, D. W. & Johnson, C. H. A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc. Natl Acad. Sci. USA 96, 151–156 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Wilson, R. Design of experiment in assessing robustness and for qualification of a cell-based potency assay. Bioanalysis 10, 737–746 (2018).

    Article  CAS  PubMed  Google Scholar 

  34. Postnikova, E. et al. Testing therapeutics in cell-based assays: factors that influence the apparent potency of drugs. PLoS One 13, e0194880 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Nierode, G., Kwon, P. S., Dordick, J. S. & Kwon, S. J. Cell-based assay design for high-content screening of drug candidates. J. Microbiol. Biotechnol. 26, 213–225 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Nies, A. T. et al. Genetics is a major determinant of expression of the human hepatic uptake transporter OATP1B1, but not of OATP1B3 and OATP2B1. Genome Med. 5, 1 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Walker, W. S. Establishment of mononuclear phagocyte cell lines. J. Immunol. Methods 174, 25–31 (1994).

    Article  CAS  PubMed  Google Scholar 

  38. Fulop, A. et al. 4-Phenyl-alpha-cyanocinnamic acid amide: screening for a negative ion matrix for MALDI-MS imaging of multiple lipid classes. Anal. Chem. 85, 9156–9163 (2013).

    Article  PubMed  Google Scholar 

  39. Gibb, S. & Strimmer, K. MALDIquant: a versatile R package for the analysis of mass spectrometry data. Bioinformatics 28, 2270–2271 (2012).

    Article  CAS  PubMed  Google Scholar 

  40. Ruh, H., Sandhoff, R., Meyer, B., Gretz, N. & Hopf, C. Quantitative characterization of tissue globotetraosylceramides in a rat model of polycystic kidney disease by primadrop sample preparation and indirect high-performance thin layer chromatography–matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry with automated data acquisition. Anal. Chem. 85, 6233–6240 (2013).

    Article  CAS  PubMed  Google Scholar 

  41. Schulz, S. et al. DMSO-enhanced MALDI MS imaging with normalization against a deuterated standard for relative quantification of dasatinib in serial mouse pharmacology studies. Anal. Bioanal. Chem. 405, 9467–9476 (2013).

    Article  CAS  PubMed  Google Scholar 

  42. Leopold, J., Popkova, Y., Engel, K. M. & Schiller, J. Recent developments of useful MALDI matrices for the mass spectrometric characterization of lipids. Biomolecules (2018).

  43. Jaskolla, T. W. & Karas, M. Compelling evidence for Lucky Survivor and gas phase protonation: the unified MALDI analyte protonation mechanism. J. Am. Soc. Mass Spectrom. 22, 976–988 (2011).

    Article  CAS  PubMed  Google Scholar 

  44. O’Rourke, M. B., Djordjevic, S. P. & Padula, M. P. The quest for improved reproducibility in MALDI mass spectrometry. Mass Spectrom. Rev. 37, 217–228 (2018).

    Article  PubMed  Google Scholar 

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The authors thank A. Dörrbaum, A. Geisel, C. Croissant and F.B.M. Reinhard for critically reviewing the manuscript and for helpful discussions. This work was funded by the German Federal Ministry of Research (BMBF) as part of the Innovation Partnership M2Aind, project SM2all (03FH8I01IA) within the framework FH-Impuls. Acquisition of the rapiflex MS was supported by the Hector Foundation II, acquisition of the solarix 7T XR by the Deutsche Forschungsgemeinschaft (Project 262133997). M.B. is supported by a Capes-Humboldt Research Fellowship for Postdoctoral Researchers [Program CAPES-HUMBOLDT N. 88881.197758/2018-01].

Author information

Authors and Affiliations



M.S.U. designed and conducted all targeted experiments of compound uptake and drug–drug interactions, and wrote the manuscript. M.B. designed and conducted the untargeted and high-resolution MS and MS/MS experiments, and wrote the manuscript. T.E. wrote R scripts and made them available to the scientific community via GitHub. C.H. designed the studies and wrote the manuscript.

Corresponding author

Correspondence to Carsten Hopf.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Laura Sanchez and Matthias Trost for their contribution to the peer review of this work.

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Key references using this protocol

Munteanu, B. et al. Anal. Chem. 86, 4642–4647 (2014):

Weigt, D. et al. Sci. Rep. 8, 11260 (2018):

Weigt, D. et al. Cell Chem. Biol. 26, 1322–1331 (2019):

Unger, M. S. et al. Anal. Chem. 92, 11851–11859 (2020):

Key data used in this protocol

Supplementary information

Reporting Summary

Supplementary Software

Readme file for R-Package: MALDIcellassay.

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Unger, M.S., Blank, M., Enzlein, T. et al. Label-free cell assays to determine compound uptake or drug action using MALDI-TOF mass spectrometry. Nat Protoc 16, 5533–5558 (2021).

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