Caspases are a family of proteases found in all metazoans, including a dozen in humans, that drive the terminal stages of apoptosis as well as other cellular remodeling and inflammatory events. Caspases are named because they are cysteine class enzymes shown to cleave after aspartate residues. In the past decade, we and others have developed unbiased proteomic methods that collectively identified~2000 native proteins cleaved during apoptosis after the signature aspartate residues. Here, we explore non-aspartate cleavage events and identify 100s of substrates cleaved after glutamate in both human and murine apoptotic samples. The extended consensus sequence patterns are virtually identical for the aspartate and glutamate cleavage sites suggesting they are cleaved by the same caspases. Detailed kinetic analyses of the dominant apoptotic executioner caspases-3 and -7 show that synthetic substrates containing DEVD↓ are cleaved only twofold faster than DEVE↓, which is well within the 500-fold range of rates that natural proteins are cut. X-ray crystallography studies confirm that the two acidic substrates bind in virtually the same way to either caspases-3 or -7 with minimal adjustments to accommodate the larger glutamate. Lastly, during apoptosis we found 121 proteins cleaved after serine residues that have been previously annotated to be phosphorylation sites. We found that caspase-3, but not caspase-7, can cleave peptides containing DEVpS↓ at only threefold slower rate than DEVD↓, but does not cleave the unphosphorylated serine peptide. There are only a handful of previously reported examples of proteins cleaved after glutamate and none after phosphorserine. Our studies reveal a much greater promiscuity for cleaving after acidic residues and the name 'cacidase' could aptly reflect this broader specificity. Human caspases are a family of 12 homologous intracellular proteases known for driving cellular state changes such as apoptosis and differentiation, as well as inflammatory responses. Caspases are cysteine-class proteases named for their signature ability to cleave after aspartate residues, or P1 is aspartate using the Schetchter and Berger notation. 1,2Synthetic peptide profiling for purified caspases show distinctive subsite preferences extending from P1 to P4 or P5. 3-5Sequence conservation and signal pathway analyses have further grouped the proteases into apoptotic initiators (−2, − 8, − 9 and − 10), apoptotic executioners (−3, − 6 and − 7), regulators of inflammation (−1, − 4, − 5 and − 12) and keratinocyte differentiation − 14).The past decade has seen a significant advancement in the use of LC-MS to define the spectrum of natural proteins cleaved by caspases in cells. [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] In addition to providing unbiased information about which proteins are cleaved, in some cases, these experiments locate the precise sites and quantify the rates of cleavage.13-17 Using the subtiligasebased N-terminomics approach, we identified more than 1700 aspartate cleavage eve...