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

Abstract 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 TNFα 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. Competing Interest Statement The authors have declared no competing interest. Abbreviations - APOA1 - Apolipoprotein A1 - ANOVA - Analysis of Variance - BCA - Bicinchoninic Acid - CD93 - Cluster of Differentiation 93 - CXCL10 - C-X-C Motif Chemokine Ligand 10 - DAVID - Database for Annotation, Visualization, and Integrated Discovery - DIA - Data-Independent Acquisition - DIA-NN - Data Independent Acquisition by Neural Networks - DPBS - Dulbecco’s Phosphate Buffered Saline - ELISA - Enzyme-Linked Immunosorbent Assay - FBS - Fetal Bovine Serum - FDR - False Discovery Rate - GO - Gene Ontology - HDL - High-Density Lipoprotein - HKMtb - Heat-Killed Mycobacterium tuberculosis - HKSA - Heat-Killed Staphylococcus aureus - HKEB - Heat-Killed Escherichia coli - IAA - Iodoacetamide - IFNγ - Interferon Gamma - IL - Interleukin - LC-MS/MS - Liquid Chromatography–Tandem Mass Spectrometry - LPS - Lipopolysaccharide - M-CSF - Macrophage Colony-Stimulating Factor - M0 - Resting Macrophage Phenotype - M1 - Pro-inflammatory Macrophage Phenotype - MS - Mass Spectrometry - nELISA - Nucleobase Enabled Localised Immunoassay with Spectral Addressing - PCA - Principal Component Analysis - PASEF - Parallel Accumulation–Serial Fragmentation - PEA - Proximity Extension Assay - PON1 - Paraoxonase 1 - RPMI - Roswell Park Memorial Institute Medium - SDS - Sodium Dodecyl Sulphate - SIMA - Signals Image Artist - SPD - Samples Per Day - STRING - Search Tool for the Retrieval of Interacting Genes/Proteins - TAK-242 - Resatorvid (TLR4 inhibitor) - TEAB - Triethylammonium Acetate Buffer - TCEP - Tris-(2-carboxyethyl)phosphine - TIMS - Trapped Ion Mobility Spectrometry - TNFα - Tumour Necrosis Factor Alpha - UniProt - Universal Protein Resource - VSN - Variance Stabilising Normalisation