a) Workflow of the MALDI-TOF DUB assay. Each of the 42 DUBs was incubated with all eight diubiquitin isomers individually (M1, K6, K11, K27, K29, K33, K48 and K63) for 60 min at 30 °C. The reaction was stopped with 2% TFA and mixed 1:1 with 0.5 μM 15N-ubiquitin which serves as an internal standard. Subsequently, the analyte is mixed with 2,5 DHAP matrix and spotted onto a 1,536 AnchorChip MALDI target (Bruker Daltonics). Data analysis is performed using FlexAnalysis (Bruker Daltonics). ( b) The MALDI-TOF DUB assay shows high sensitivity. Zoomed area (8,520–8,720 m/ z) of MALDI-TOF MS spectra for ubiquitin (Ubi) and 15N-ubiquitin, in the presence of K11-linked diubiquitin are depicted. The limit of detection was determined as 2 fmol of ubiquitin on the target (in the presence of 42 fmol of 15N-ubiquitin and 146 fmol of K11-linked diubiquitin). Presence of the doubly charged diubiquitin (diubiquitin [M+2H] 2+) does not compromise identification of the singly charged ubiquitin (see also Supplementary Fig. 2). ( c) Linearity and reproducibility of the MALDI-TOF DUB assay. Scatter plot of different concentrations of ubiquitin (10–10,000 nM) shows high linearity over about three orders of magnitude. Interday reproducibility was very high ( Supplementary Table 1). Error bars represent s.d. of measurements. a.u., arbitrary unit; intens., intensity.