Figure 2 : Characterizing the linkage specificity of DUBs.

From: Screening of DUB activity and specificity by MALDI-TOF mass spectrometry

Figure 2

Increasing concentrations (0.02–200 ng μl−1) of DUBs were incubated in triplicate with 1.46 μM of diubiquitin of each linkage type (M1, K6, K11, K29, K33, K48, K63 from Boston Biochem, K27 in-house produced) for 60 min at 30 °C and analysed by the MALDI-TOF DUB assay. The amount of monoubiquitin formed by this reaction was determined by MALDI-TOF MS and used to establish the DUB activity for individual diubiquitin isomers which is shown in a gradient of white (0%) to dark red (100%). The data show that DUBs can be grouped into enzymes cleaving specifically one linkage type (group 1), few linkage types (group 2), unspecific (group 3) or inactive enzymes (group 4). For DUB characterization, see Supplementary Figs 3 and 8.