Author:

Keywords:

Science & Technology, Life Sciences & Biomedicine, Biochemical Research Methods, Biochemistry & Molecular Biology, N-TERMINAL PEPTIDES, CATHEPSIN-E, EXPRESSION, APOPTOSIS, PRODUCTS, SITES, SILAC, TOOL, Amino Acid Sequence, Animals, Caspase 3, Cathepsin D, Cathepsin E, Humans, Hydrolysis, Mice, Molecular Sequence Data, Proteomics, Substrate Specificity

Abstract:

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.