Figure 1 : The MALDI-TOF DUB assay.

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

Figure 1

(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.