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. To identify existing and novel Calpain-2 substrates, 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 substrates that may be cleaved directly by Calpain-2 or indirectly by downstream proteases. The direct cleavage of selected substrates by Calpain-2 was confirmed using in vitro assays. Finally, metabolomics analysis 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.