TY - JOUR
T1 - Mass spectrometry analysis to identify ubiquitylation of eyfp-tagged cenp-a (Eyfp-cenp-a)
AU - Niikura, Yohei
AU - Fang, Lei
AU - Kitagawa, Risa
AU - Li, Peizhao
AU - Xi, Yao
AU - You, Ju
AU - Guo, Yan
AU - Kitagawa, Katsumi
N1 - Funding Information:
We thank Chao-Jun Li at the Model Animal Research Center, Nanjing University for mass spectrometry analysis. We thank Yanmini Dalal, Tatsuo Fukagawa, and current researchers at the Model Animal Research Center, Nanjing University and Greehey Children?s Cancer Research Institute for their helpful discussion, experimental guidance, and reagents. We thank Don W. Cleveland, Daniele Fachinetti, Yanmini Dalal, Minh Bui, Gustavo W. Leone, John Thompson, Lawrence S. Kirschner, Amruta Ashtekar, Ben E. Black, Glennis A. Logsdon, Kenji Tago, and Dawn S. Chandler for their generous gifts of reagents. Y.N. was supported by Jiangsu Province ??Double-First-Class?? Construction Fund, Jiangsu Province Natural Science Fund (SBK2019021248), Jiangsu Province 16th Six Big Talent Peaks Fund (TD-SWYY-001), Jiangsu Province ?Foreign Expert Hundred Talents Program? Fund (SBK2019010048), and National Natural Science Foundation in China (31970665). This study was partly supported by NCI grant R21 CA205659.
Funding Information:
We thank Yanmini Dalal, Tatsuo Fukagawa, and current researchers at the Model Animal Research Center, Nanjing University and Greehey Children’s Cancer Research Institute for their helpful discussion, experimental guidance, and reagents. We thank Don W. Cleveland, Daniele Fachinetti, Yanmini Dalal, Minh Bui, Gustavo W. Leone, John Thompson, Lawrence S. Kirschner, Amruta Ashtekar, Ben E. Black, Glennis A. Logsdon, Kenji Tago, and Dawn S. Chandler for their generous gifts of reagents. Y.N. was supported by Jiangsu Province ‘‘Double-First-Class’’ Construction Fund, Jiangsu Province Natural Science Fund (SBK2019021248), Jiangsu Province 16th Six Big Talent Peaks Fund (TD-SWYY-001), Jiangsu Province “Foreign Expert Hundred Talents Program” Fund (SBK2019010048), and National Natural Science Foundation in China (31970665). This study was partly supported by NCI grant R21 CA205659.
Publisher Copyright:
© 2020, Journal of Visualized Experiments. All rights reserved.
PY - 2020/6
Y1 - 2020/6
N2 - 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.
AB - 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.
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U2 - 10.3791/61138
DO - 10.3791/61138
M3 - Article
C2 - 32597847
AN - SCOPUS:85087435304
VL - 2020
SP - 1
EP - 21
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
SN - 1940-087X
IS - 160
M1 - e61138
ER -