Mass spectrometry analysis to identify ubiquitylation of eyfp-tagged cenp-a (Eyfp-cenp-a)

Yohei Niikura, Lei Fang, Risa Kitagawa, Peizhao Li, Yao Xi, Ju You, Yan Guo, Katsumi Kitagawa

Research output: Contribution to journalArticle

Abstract

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle. The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Original languageEnglish (US)
Article numbere61138
Pages (from-to)1-21
Number of pages21
JournalJournal of Visualized Experiments
Volume2020
Issue number160
DOIs
StatePublished - Jun 2020

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

Fingerprint Dive into the research topics of 'Mass spectrometry analysis to identify ubiquitylation of eyfp-tagged cenp-a (Eyfp-cenp-a)'. Together they form a unique fingerprint.

  • Cite this