Ubiquitin chain analysis by parallel reaction monitoring

Joseph Longworth, Marta L. Mendes, Gunnar Dittmar*

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review


Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The protocol outlined here takes advantage of the di-glycine (-GG) modification left after the tryptic digestion of ubiquitin incorporated in a chain. By quantifying these topology-characteristic peptides the relative abundance of each ubiquitin chain topology can be determined. The steps required to quantify these peptides by a parallel reaction monitoring experiment are reported taking into consideration the stabilization of ubiquitin chains. Preparation of heavy controls, cell lysis, and digestion are described along with the appropriate mass spectrometer setup and data analysis workflow. An example data set with perturbations in ubiquitin topology is presented, accompanied by examples of how optimization of the protocol can affect results. By following the steps outlined, a user will be able to perform a global assessment of the ubiquitin topology landscape within their biological context.

Original languageEnglish
Article numbere60702
Pages (from-to)1-16
Number of pages16
JournalJournal of Visualized Experiments
Issue number160
Publication statusPublished - Jun 2020


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