biostudies-literatureUnknown9(1)Tsuchiya HProtein ubiquitylation regulates diverse cellular processes via distinct ubiquitin chains that differ by linkage type and length. However, a comprehensive method for measuring these properties has not been developed. Here we describe a method for assessing the length of substrate-attached polyubiquitin chains, "ubiquitin chain protection from trypsinization (Ub-ProT)." Using Ub-ProT, we found that most ubiquitylated substrates in yeast-soluble lysate are attached to chains of up to seven ubiquitin molecules. Inactivation of the ubiquitin-selective chaperone Cdc48 caused a dramatic increase in chain lengths on substrate proteins, suggesting that Cdc48 complex terminates chain elongation by substrate extraction. In mammalian cells, we found that ligand-activated epidermal growth factor recepNature communications524https://www.ebi.ac.uk/biostudies/studies/S-EPMC5802829biostudies-literatureUb-ProT reveals global length and composition of protein ubiquitylation in cells.PMC5802829Burana DSaeki YKomada MOhtake FArai NKaiho ATanaka KTsuchiya HfalseUb-ProT reveals global length and composition of protein ubiquitylation in cells.Protein ubiquitylation regulates diverse cellular processes via distinct ubiquitin chains that differ by linkage type and length. However, a comprehensive method for measuring these properties has not been developed. Here we describe a method for assessing the length of substrate-attached polyubiquitin chains, "ubiquitin chain protection from trypsinization (Ub-ProT)." Using Ub-ProT, we found that most ubiquitylated substrates in yeast-soluble lysate are attached to chains of up to seven ubiquitin molecules. Inactivation of the ubiquitin-selective chaperone Cdc48 caused a dramatic increase in chain lengths on substrate proteins, suggesting that Cdc48 complex terminates chain elongation by substrate extraction. In mammalian cells, we found that ligand-activated epidermal growth factor recep2018-01-01T00:00:00Z2018 Feb2026-05-03T18:07:10.238Z2026-04-07T19:32:20.451ZS-EPMC58028292941040110.1038/s41467-018-02869-x