Isolation and Mass Spectrometry Identification of K48 and K63 Ubiquitin Proteome Using Chain-Specific Nanobodies

Maria Gonzalez-Santamarta, Laurie Ceccato, Ana Sofia Carvalho, Jean Christophe Rain, Rune Matthiesen, Manuel S. Rodriguez

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

1 Citation (Scopus)

Abstract

Protein ubiquitylation is an essential mechanism regulating almost all cellular functions in eukaryotes. The understanding of the role of distinct ubiquitin chains in different cellular processes is essential to identify biomarkers for disease diagnosis and prognosis but also to open new therapeutic possibilities. The high complexity of ubiquitin chains complicates this analysis, and multiple strategies have been developed over the last decades. Here, we report a protocol for the isolation and identification of K48 and K63 ubiquitin chains using chain-specific nanobodies associated to mass spectrometry. Different steps were optimized to increase the purification yield and reduce the binding on nonspecific proteins. The resulting protocol allows the enrichment of ubiquitin chain-specific targets from mammalian cells.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press
Pages125-136
Number of pages12
DOIs
Publication statusPublished - 2023

Publication series

NameMethods in Molecular Biology
Volume2602
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Isolation
  • Mass spectrometry
  • Nanobodies
  • Posttranslational modifications
  • Proteome
  • Ubiquitin

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