We present here a novel proteomics design for systematic identification of 
protease cleavage events by quantitative N-terminal proteomics, circumventing 
the need for time-consuming manual validation. We bypass the singleton detection 
problem of protease-generated neo-N-terminal peptides by introducing 
differential isotopic proteome labeling such that these substrate reporter 
peptides are readily distinguished from all other N-terminal peptides. Our 
approach was validated using the canonical human caspase-3 protease and further 
applied to mouse cathepsin D and E substrate processing in a mouse dendritic 
cell proteome, identifying the largest set of protein protease substrates ever 
reported and gaining novel insight into substrate specificity differences of 
these cathepsins.