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Proteasomal proteolysis relies on the activity of six catalytically active proteasomal subunits (beta1, beta2, beta5, beta1i, beta2i and beta5i). Applying a functional proteomics approach, we used a recently developed activity-based, cell-permeable proteasome-specific probe that for the first time allows differential visualization of individual active proteasomal subunits in intact primary cells. In primary leukemia samples, we observed remarkable variability in the amounts of active beta1/1i-, beta2/2i- and beta5/5i-type of subunits, contrasting with their constant protein expression. Bortezomib inhibited beta5- and beta1-type, but to a lesser extend beta2-type of subunits in live primary cells in vitro and in vivo. When we adapted the bortezomib-sensitive human acute myeloid leukemia cell line HL-60 to bortezomib 40 nM (HL-60a), proteasomal activity profiling revealed an upregulation of active subunits, and residual beta1/beta5-type of activity could be visualized in the presence of bortezomib 20 nM, in contrast to control cells. In a panel of cell lines from hematologic malignancies, the ratio between beta2-type and (beta1 + beta5)-type of active proteasomal polypeptides mirrored different degrees of bortezomib sensitivity. We thus conclude that the proteasomal activity profile varies in primary leukemia cells, and that the pattern of proteasomal subunit activity influences the sensitivity of hematologic malignancies toward bortezomib.