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 signaling, with its dysregulation leading to widespread physiological dysfunction and disease. Immunoassay formats that utilize 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 robustly identified over 900 protein groups in under 15 min of acquisition time, delivering exceptional reproducibility across a large sample set. We applied this approach to profile proinflammatory 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 h 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 IL-6 initiating downstream signaling cascades that resulted in the delayed secretion of chemokines such as CXCL10 and CCL8. Collectively, these findings establish a robust, scalable platform for global characterization 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.