MALDI-TOF mass spectrometry and proteomics as phenotypic screening tools for anti-inflammatory drugs

Trost, M.; Peltier-Heap, R. E.; Müller, L.; Bateman, S.; Haslam, C.; Dawson, T.; Vlachou, D. F.; Rosa-Roseberry, E.; Booty, L. M.; Tayler, C. L.; Frey, A. M.; Annan, R. S.; Leveridge, M.; Duenas, M. E.

doi:10.64898/2025.12.01.691706

bioRxiv · 10.64898/2025.12.01.691706

MALDI-TOF mass spectrometry and proteomics as phenotypic screening tools for anti-inflammatory drugs

Trost, M.·Peltier-Heap, R. E.·Müller, L.·Bateman, S.·Haslam, C.·Dawson, T.·Vlachou, D. F.·Rosa-Roseberry, E.·Booty, L. M.·Tayler, C. L.·Frey, A. M.·Annan, R. S.·Leveridge, M.·Duenas, M. E.

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Abstract

Phenotypic screening is a powerful technology to discover drug candidates in physiologically relevant systems without prior knowledge of molecular targets; however, mass spectrometry (MS) remains underutilised as readout strategy. In this proof-of-concept study, we developed and evaluated two complementary MS-based phenotypic screening approaches to identify anti-inflammatory compounds in human induced pluripotent stem cell-derived macrophages and compared them to a conventional targeted cytokine profiling assay. First, we established a novel MALDI-TOF MS fingerprinting strategy that effectively distinguished macrophage phenotypes, identified phenotype-specific biomarkers, and maintained high-throughput capabilities while reducing cost. Secondly, we performed an in-depth LC-MS proteomic analysis using low cell input on an Evosep-timsTOF HT setup, providing rich molecular detail. Both MS-based approaches demonstrated large comparability with the cytokine assay, with a large proportion of hits overlapping. Notably, the proteomics workflow uniquely enabled deeper insight into inflammation pathway engagement, off-target effects, compound potency, and cytotoxicity. Together, these findings highlight the potential of MS-driven phenotypic screening to enhance early drug discovery by enabling efficient, informative, and cost-effective hit selection. O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/691706v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@c83cb7org.highwire.dtl.DTLVardef@a14d4org.highwire.dtl.DTLVardef@1dda6d7org.highwire.dtl.DTLVardef@f47564_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Trost, M., Peltier-Heap, R. E., Müller, L., Bateman, S., Haslam, C., Dawson, T., Vlachou, D. F., Rosa-Roseberry, E., Booty, L. M., Tayler, C. L., Frey, A. M., Annan, R. S., Leveridge, M., Duenas, M. E.. 2025-12-03. MALDI-TOF mass spectrometry and proteomics as phenotypic screening tools for anti-inflammatory drugs. https://doi.org/10.64898/2025.12.01.691706

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