Extended Data Fig. 9: Cleavage efficiency of phospho-polyubiquitin chains and MS analysis of parkin assembly reactions.
From: Insights into ubiquitin chain architecture using Ub-clipping
a, In vitro assemblies of polyubiquitin from ubiquitin and Ser65 phosphoubiquitin using cIAP1 and UBE2D3 as previously described11, and their cleavage by Lbpro. Cleavage efficiency was visualized by Coomassie staining. Experiments were independently performed in duplicate with similar results. b, Samples from a visualized by anti-ubiquitin western blotting. Experiments were independently performed in duplicate with similar results. c, Polyubiquitin assembled by Ser65-phosphorylated human parkin (pParkin) with UBE2L3 was purified by GST–TUBE pull-downs. Assembly reactions were performed in the absence (−) and presence (+) of 10% total phosphoubiquitin. Experiments were performed independently in triplicate. d, TUBE-purified parkin reactions from c were analysed by AQUA MS to determine chain-linkage composition. A representative example of independent experiments performed in triplicate is shown. e, Lbpro-treated parkin assembly reactions were analysed by intact MS and spectra deconvolution. A small phosphoubiquitin contamination is present owing to residual PINK1 kinase in the assembly reactions. In these assays, parkin predominantly assembles monoubiquitin and short chains. Intact MS analysis was performed independently in duplicate. f, To detect a non-ubiquitin substrate with multiple GlyGly modifications by intact MS, in vitro assemblies were performed as per e in the absence of phosphoubiquitin, and with a higher UBE2L3 concentration to facilitate the analysis of UBE2L3. Lbpro-treated assemblies were analysed by LC–MS, revealing up to four GlyGly modifications on a single UBE2L3 molecule. Inset, the distribution of GlyGly-modified UBE2L3 in the +21 charge state. The experiment was performed independently in triplicate.
