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. 2021 Oct 26;12:6173. doi: 10.1038/s41467-021-26427-0

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a TetraUb-Cyclin B1-NT was incubated with purified 20S or 26S proteasomes at 1:150 (proteasome:substrate) molar ratio at 37 °C for indicated time periods. The reaction mixture was resolved by Tris-Tricine PAGE and stained with Coomassie. Brotezomib was added to reactions to inhibit the proteasome activity. b MonoUb- or DiUb-Cyclin B1-NT were incubated at 37 °C with purified 20S or 26S proteasomes at 1:150 (proteasome:substrate) molar ratio for indicated time periods and separated in Tris-Tricine PAGE followed by Coomassie staining. c The illustration represents the ubiquitin peptides obtained from TetraUb-Cyclin B1-NT proteolyzed by the 20S proteasome detected by MS/MS analysis overlaid on the primary sequences of the ubiquitin units in the chain. Purple-colored residues represent the Flag-tag sequence on the distal ubiquitin unit, while green represents the Myc-tag sequence on the proximal ubiquitin. d The illustration represents the branched peptides at the isopeptide linkage of the C-terminus of ubiquitin to the K64 residue of cyclin B1 identified by non-tryptic MS/MS of reaction products. Three major types of branched peptides at K64 of Cyclin B1 were detected with variants of ubiquitin remnants: K64—GG, K64—GGR, and K64—GGRL. e MS/MS spectra of one of the detected branched peptides ARLPLPKE with a GGR branch on K. Source data are provided as a Source data file.