N-terminomics and proteomics analysis of Calpain-2 reveal key proteolytic processing of metabolic and cell adhesion proteins

Aberrant levels of the cysteine protease Calpain-2 have been linked to neurodegeneration, inflammation, and cancer, yet our understanding of this protease and its substrates remains limited. Systematic studies to identify Calpain-2 substrates have been largely confined to peptide libraries or in vitro studies, which fail to represent physiological cellular conditions and physiologically relevant substrates. In addition to Calpain-2, Calpain-1 is also ubiquitously present in cells, making it challenging to understand the distinct contributions of Calpain-1 and Calpain-2 in cells and the difference between their substrates’ repertoires. We used a genetic approach to knockout Calpain-2 in the THP-1 human monocyte-like cells, followed by proteomic and N-terminomic/TAILS mass spectrometry approaches to identify Calpain-2 substrates. We identified 51 new putative Calpain-2 substrates. The direct cleavage of selected substrates by Calpain-2 was confirmed using in vitro assays. Metabolomics was performed and identified a role for Calpain-2 in the regulation of pyrimidine and glutathione metabolism. Our unbiased and quantitative mass spectrometry analytical pipeline provides new evidence on the physiological functions of Calpain-2 and its newly identified substrates in THP-1 cells.