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. 2021 Jan 21;118(4):e2006947118. doi: 10.1073/pnas.2006947118

Fig. 1.

Fig. 1.

MALDI-TOF–based methodology for profiling DUB isopeptidase/esterase activity. (A) DUBs are incubated either with ubiquitin-lysine (Ub-Lys) or ubiquitin-threonine (Ub-Thr). (B) Reactions are quenched by addition of TFA (2%), spotted on an AnchorChip 1536 target and analyzed by MALDI-TOF mass spectrometry. DUB isopeptidase or esterase activity releases free lysine or threonine and generates native Ub (8565.76 Da) in a time-dependent manner. Ion intensity ratios for peaks corresponding to Ub-Lys (8731.8 Da)/Ub-Thr (8709.6 Da) versus Ub (8565.76 Da) are used for qualitative assessment of % cleavage.