Plate-based ISD-SPE enables dual proteome-secretome concentration-response profiling of TLR signalling in iPSC-derived macrophages

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Abstract Protein secretion represents a key functional output of cellular signalling, capturing dynamic responses to stimulation and pharmacological perturbation that shape immune behaviour. In macrophages, activation of Toll-like receptors (TLRs) drives tightly regulated secretion programmes that mediate inflammatory responses and provide a biologically meaningful readout of pathway activity. Whilst mass spectrometry (MS)-based secretomics enables unbiased profiling of these processes, broader application in drug discovery remains constrained by sample preparation workflows that limit scalability. Here, we describe a plate-based in-solution digestion and solid-phase extraction (ISD-SPE) workflow that enables 96-well processing of conditioned media for integrated proteome and secretome analysis from the same sample well. Benchmarking against a precipitation-based approach demonstrated comparable proteomic depth with improved quantitative reproducibility and robust performance across multiple plates. Coupled with dia-PASEF acquisition, this workflow enabled in-depth profiling of macrophage responses to TLR activation, resolving receptor-specific secretory programmes following TLR3, TLR4 and TLR7/8 activation. Extension of the approach to concentration-response studies enabled quantitative characterisation of pharmacological perturbation across intracellular and extracellular protein landscapes, revealing both shared and compartment-specific responses to TLR inhibition, as well as differences in apparent potency linked to secretion dynamics. Together, this workflow provides a scalable strategy for integrated analysis of intracellular signalling and downstream protein secretion, enabling systems-level characterisation of inflammatory responses and compound mechanisms of action.

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