Ubiquitin Chain Analysis by Parallel Reaction Monitoring.
J Vis Exp
; (160)2020 06 17.
Article
in En
| MEDLINE
| ID: mdl-32628175
ABSTRACT
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Mass Spectrometry
/
Ubiquitin
Limits:
Animals
/
Humans
Language:
En
Journal:
J Vis Exp
Year:
2020
Type:
Article