a, Experiments were performed with doxycycline-inducible HeLa Flp-In T-REx cells expressing parkin proteins (a gift from A. Ordureau and W. Harper, Harvard). Cells were treated with 0.1 μg ml−1 doxycycline for 16 h and parkin expression was monitored by anti-parkin western blots. Western blots were performed on one of three independent experiments. b, TUBE enrichment of polyubiquitin from a after CCCP treatment. Also see Fig. 4a. Western blots were performed on one of three independent experiments. c, Top, AQUA analysis for total ubiquitin, ubiquitin chains, and phosphoubiquitin using TUBE pull-downs from CCCP-treated parkin-overexpressing cell lines (see a, b, Fig. 4a). TUBE-purified ubiquitin chains were treated with Lbpro∗, separated by SDS–PAGE, and the monoubiquitin band excised and subjected to AQUA MS analysis. Bottom, the percentages calculated from the top panel. Values correspond to the mean of independent experiments performed in triplicate (n = 3). Error values represent s.d. from the mean. d, Quantification of ubiquitin species from the parkin(S65A) cell line, as in Fig. 4d. Centre values correspond to the mean of independent experiments performed in triplicate (n = 3). Error values represent s.d. from the mean. e, Sodium carbonate (Na 2 CO 3 ) treatment of mitochondria and Lbpro cleavage assays. Addition of Na 2 CO 3 releases peripheral mitochondria membrane proteins and unconjugated free monoubiquitin. Untreated and Na 2 CO 3 -treated mitochondria were digested with Lbpro for 2.5 h at 37 °C. After incubation for 2.5 h, the supernatant (S) and pellet (P) were analysed by anti-ubiquitin western blots. Without Na 2 CO 3 , incubation at 37 °C releases ubiquitin from mitochondria into the supernatant, presumably owing to residual DUB activity. Western blots were performed on one of three independent experiments. f, Left and middle, identification of non-ubiquitinated phosphoubiquitin by intact MS analysis from Lbpro-treated samples in e. The isotopic distribution of the charge state z = +12 is shown for parkin wild-type and parkin(C431S) cell lines. Right, the fold-increase in phosphoubiquitin comparing parkin(C431S) and wild-type cell lines, as measured by spectra deconvolution. A considerable amount of phosphoubiquitin (10% of the total ubiquitin) can be detected with this method in cells expressing inactive parkin. Centre values correspond to the mean of independent experiments performed in triplicate (n = 3). g, To exclude the possibility of contaminating phosphatase activity during incubation with Lbpro∗, sodium carbonate-extracted mitochondrial samples were treated as in Fig. 4h and spiked with 15N-labelled phosphoubiquitin, and formation of 15N-labelled unphosphorylated ubiquitin was monitored. As indicated, no detectable formation of a 15N-labelled unphosphorylated ubiquitin species was observed after overnight incubation with Lbpro∗. Experiments were performed in biological triplicate. h, As in Extended Data Fig. 8e, the data from Fig. 4h is applied to a pool of 20 ubiquitin molecules, in which eight are unmodified, five are GlyGly-modified, one is double-GlyGly modified, and six are phosphorylated. This simplified system would allow for a distribution of chains as depicted schematically, and would indicate that chains on mitochondria are short and phosphorylated on the tips of the chain. Recent site occupancy mapping—performed in the same cell system32—revealed abundant ubiquitination sites in particular on VDAC proteins, enabling us to model the ubiquitin coat during mitophagy, as depicted. Centre values correspond to the mean of independent experiments performed in triplicate. Error values represent s.d. from the mean. Source data