Sensitive Determination of Proteolytic Proteoforms in Limited Microscale Proteome Samples

Protein N termini reveal fundamental regulatory mechanisms and their perturbation in disease. Regulatory proteolysis is often spatially and temporally confined, thus accessible only in minimal specimen incompatible with established protocols. We developed a robust, sensitive, scalable and automatable method for system-wide identification of thousands of N termini from minute samples. Applications revealed distinct N-terminal profiles in sorted immune cells and mitochondria from pediatric cancer patient cells, protease substrates in Arabidopsis seedlings and effects of chemotherapy on proteolytic proteoforms in clinical liquid biopsies.