Deep protease profiling deconvolutes protease signatures released from activated neutrophils

Dysregulated protease activity drives the progression of cancer, cardiovascular, and inflammatory diseases. Current substrate-based detection methods lack specificity, breadth, and require prior knowledge of protease content. We have advanced deep protease profiling, through differential enrichment, substrate motif, and deconvolution analyses, to provide comprehensive, unbiased, and scalable characterizations of protease activity. Phage libraries were screened against neutrophil serine proteases or activated neutrophils, and high-throughput sequencing determined cleaved peptides. Cathepsin G, neutrophil elastase, and proteinase 3 (PR3) cleaved 5,985, 5,325, and 1,285 peptides, with substrate recognition motifs that align with natural substrates. Activated neutrophil supernatants cleaved 5,166 peptides, and inhibition of serine proteases caused 95% reduction in signal. Deconvolution algorithms identified protease signatures for elastase, cathepsin G, and PR3 proportional to their levels in neutrophils. This approach enables systematic functional analysis of proteolysis, adaptable across proteases, samples, and scenarios to advance disease understanding, diagnosis, and management.