Biology subjects

Tayler, C. L.

Publications and source records attributed to Tayler, C. L..

4 recordsLinked to original sources

bioRxiv2026

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

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.

immunology↗

bioRxiv2026

dia-PASEF Enables Rapid Profiling of the Human Secretome for Deeper Insights into Cellular Dynamics and Inflammatory Mechanisms

Protein secretion is a fundamental mechanism for cellular coordination and signalling, with its dysregulation leading to widespread physiological dysfunction and disease. Immunoassay formats that utilise secondary antibody readouts are the current gold standard for measuring secreted proteins, offering high specificity and sensitivity, but relying on predefined protein panels that constrain the discovery of novel biology. We present a scalable mass spectrometry-based workflow that combines data-independent acquisition with ion mobility and parallel fragmentation to deliver rapid, global profiling of the secretome. Using a translationally relevant human iPSC-derived macrophage model, our approach identified over 1200 proteins in under 15 minutes of acquisition time, delivering exceptional reproducibility across a large sample set. We applied this approach to profile pro-inflammatory phenotypes, confirming robust identification of key cytokines and chemokines whilst revealing non-canonical immune responses absent from both targeted panels and the intracellular proteome. In particular, we identified a unique cholesterol efflux signature, marked by the secretion of APOA1 and PON1, in response to Mycobacterium Tuberculosis, consistent with the metabolic reprogramming that takes place during infection. Furthermore, temporal profiling of macrophage responses to lipopolysaccharide over 24 hours resolved dynamic secretion trajectories that distinguish between acute and chronic inflammatory states. The extended time period facilitated the observation of distinct cytokine-dependent secretion phenotypes, with early secretion of TNF and IL6 initiating downstream signalling cascades that resulted in the delayed secretion of chemokines such as CXCL10 and CCL8. Collectively, these findings establish a robust, scalable platform for global characterisation of secretory networks. Beyond macrophage biology, this workflow offers broad utility for biomarker discovery, mechanistic studies of disease progression and evaluation of new therapeutic interventions, providing a powerful tool for advancing precision medicine.

immunology↗

bioRxiv2025

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

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

biochemistry↗

bioRxiv2025

Discovery of an orally available potent ER aminopeptidase 1 (ERAP1) inhibitor that enhances anti-tumor responses and limits inflammatory autoimmunity in vivo.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that can regulate immune responses primarily by proteolytically processing peptides before loading and presentation on the cell surface by major histocompatibility class I molecules (MHC-I). ERAP1 activity can either reduce the immunogenicity of cancer cells by over-trimming cancer-associated antigenic peptides or contribute to autoimmunity by generating self-antigenic peptides. As a result, ERAP1 inhibition has emerged as a tractable approach for cancer immunotherapy and specific classes of autoimmunity. Here, we describe the discovery, after hit-to-lead optimization, of a potent and selective ERAP1 inhibitor based on the pyrrolidine 3-carboxylic acid scaffold that targets the regulatory allosteric site. The compound has favourable in vivo pharmacokinetics, including oral bioavailability, and can regulate the immunopeptidome of cancer cells and enhance cancer cell antigenicity in vivo in a dose-dependent manner, controlling tumor growth. In addition, when administered in the murine collagen-induced arthritis model, it does not induce any exacerbation of autoimmune responses but rather results in a dose-dependent therapeutic benefit. Our results demonstrate that ERAP1 inhibition can constitute a tractable approach to modulating immune responses for therapeutic applications, providing mechanistic insight and a valuable lead and in vivo tool for further drug development efforts and for interrogating ERAP1 biology.

immunology↗