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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.

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


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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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