Multiplexed protease activity profiling for host response-based Sepsis diagnosis.

Human proteases play major roles in various pathological conditions, including dysregulated immune responses in sepsis, making them strong candidates for developing diagnostic markers. Despite this potential, the process of developing protease-based diagnostic tools has remained slow due to significant technical barriers associated with measuring protease activity, mainly stemming from the vast diversity of human proteases and the lack of substrate specificity, which complicates the interpretation of activity profiles. In this work, we advanced the current state of assay development by engineering substrate molecules and implementing an analytical approach based on mass spectrometry. Specifically, we developed a novel approach to chemically modify protease substrates for human neutrophil elastase (HNE) and matrix metalloproteinases (MMPs) to enhance specificity and generate unique mass-to-charge signatures, allowing the deconvolution of protease activities. We then integrated the modified substrates into a mass spectrometry-based platform for quantifying multiple protease activities within a single analytical run. We applied the assay to spared plasma samples and demonstrated that the assay detects distinct protease activity profiles. We further demonstrated that the assay achieved a diagnostic sensitivity of 88% and specificity of 87% for sepsis detection within a three-hour timeframe. ONE SENTENCE SUMMARY: We developed a novel multiplexed protease activity profiling approach to quantify immune responses and improve sepsis diagnosis.