DDI2 has been shown to cleave and activate the ER-bound transcription factor TCF11/NRF1 (NFE2L1), a master regulator of proteostasis maintenance in mammalian cells, to ensure expression of rescue factors including proteasome subunits upon proteotoxic stress. Recent studies have identified DDI1-like proteases as endoproteases that cleave polyubiquitinated substrates. The unusual nature of these aspartic proteases is further supported by the fact that they participate in DNA repair. Despite all these findings, nothing is known about the physiological role and functional complexity of DDI2 function in cells. We therefore generated a CRISR/Cas9 knock-out of DDI2 in the human endothelial cell line Ea.Hy926 and compared the total proteome of the parental cell line with that of the DDI2 knock-out line by mass spectrometry.