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&#8217;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 &#8216;&#8216;Double- First-Class&#8217;&#8217; Construction Fund, Jiangsu Province Natural Science Fund (SBK2019021248), Jiangsu Province 16th Six Big Talent Peaks Fund (TD-SWYY-001), Jiangsu Province &#8220;Foreign Expert Hundred Talents Program&#8221; Fund (SBK2019010048), and National Natural Science Foundation in China (31970665). This study was partly supported by NCI grant R21 CA205659.

<ol>
	<li>Dunleavy, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HJURP+is+a+cell-cycle-dependent+maintenance+and+deposition+factor+of+CENP-A+at+centromeres.">HJURP is a cell-cycle-dependent maintenance and deposition factor of CENP-A at centromeres.</a> Cell. 137 (3), 485-497 (2009).</li><li>Foltz, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centromere-specific+assembly+of+CENP-a+nucleosomes+is+mediated+by+HJURP.">Centromere-specific assembly of CENP-a nucleosomes is mediated by HJURP.</a> Cell. 137 (3), 472-484 (2009).</li><li>Bernad, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+HJURP+and+condensin+II+are+required+for+CENP-A+assembly.">Xenopus HJURP and condensin II are required for CENP-A assembly.</a> J Cell Biol. 192 (4), 569-582 (2011).</li><li>Niikura, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CENP-A+K124+Ubiquitylation+Is+Required+for+CENP-A+Deposition+at+the+Centromere.">CENP-A K124 Ubiquitylation Is Required for CENP-A Deposition at the Centromere.</a> Developmental Cell. 32 (5), 589-603 (2015).</li><li>Niikura, Y., Kitagawa, R., Kitagawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CENP-A+Ubiquitylation+Is+Inherited+through+Dimerization+between+Cell+Divisions.">CENP-A Ubiquitylation Is Inherited through Dimerization between Cell Divisions.</a> Cell Reports. 15 (1), 61-76 (2016).</li><li>Fachinetti, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CENP-A+Modifications+on+Ser68+and+Lys124+Are+Dispensable+for+Establishment,+Maintenance,+and+Long-Term+Function+of+Human+Centromeres.">CENP-A Modifications on Ser68 and Lys124 Are Dispensable for Establishment, Maintenance, and Long-Term Function of Human Centromeres.</a> Developmental Cell. 40 (1), 104-113 (2017).</li><li>Niikura, Y., Kitagawa, R., Kitagawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CENP-A+Ubiquitylation+Is+Required+for+CENP-A+Deposition+at+the+Centromere.">CENP-A Ubiquitylation Is Required for CENP-A Deposition at the Centromere.</a> Developmental Cell. 40 (1), 7-8 (2017).</li><li>Niikura, Y., Kitagawa, R., Fang, L., Kitagawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CENP-A+Ubiquitylation+Is+Indispensable+to+Cell+Viability.">CENP-A Ubiquitylation Is Indispensable to Cell Viability.</a> Developmental Cell. 50 (6), 683-689 (2019).</li><li>Srivastava, S., Foltz, D. 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The authors declare no competing interests.

Here we described methods of mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that the EYFP tagging induces ubiquitylation at a different lysine in the CENP-A K124R mutant protein8. In our results, we successfully identified ubiquitylation on lysine 306 (K306) in EYFP-CENP-A K124R, that is corresponding to lysine 56 (K56) in CENP-A through mass spectrometry analysis. The mass spectrometry analysis described here is a mimic method as we previously identif...

In most eukaryotes, spindle microtubules must attach to a single region of each chromosome, termed as centromere. The kinetochore is a complex of proteins that are located at the centromere. Studying the timing of centromere and kinetochore protein&#8217;s movements and the structure of kinetochores and centromeres is important for understanding chromosome instability (CIN) and cancer progression. The key questions are how the chromosomal location and function of a centromere (i.e., centromere identity) are determined and how they participate in accurate chromosome segregation. In most species, the presence of a special nucleosome containing a specific histone-like protein called CENP-A defines the centromere identity. Therefore, it is proposed that CENP-A is the non-DNA indicator (epigenetic mark) of centromere identity. It is important to elucidate the mechanism of how CENP-A defines the centromere identity in humans.
The Holliday junction recognition protein (HJURP) is the CENP-A-specific chaperon which deposits CENP-A in centromeric nucleosomes1,2,3. We have previously reported that the CUL4A-RBX1-COPS8 E3 ligase is required for CENP-A ubiquitylation on lysine 124 (K124) and centromere localization4. Also, our results showed that the centromere recruitment of newly synthesized CENP-A requires pre-existing ubiquitylated CENP-A5. Thus, a model was provided suggesting that CENP-A ubiquitylation is inherited through dimerization between cell divisions.
In contrast to our findings and those of Yu et al., negative results regarding the CENP-A and its centromeric localization were recently published6. The article claimed that CENP-A modifications on lysine 124 (K124) are dispensable for the establishment, maintenance, and long-term function of human centromeres, based on their negative results showing that the mutation of K124R did not affect CENP-A centromere localization neither cell viability6. However, there is enough room for debate in their results and conclusions, and we have already described what problem there could be in their previous publication7. Attention should be paid that they fused proteins with CENP-A, which have much larger molecular weights than the size of endogenous CENP-A: e.g., they fused ~30 kDa enhanced yellow fluorescent protein (EYFP) to ~16 kDa CENP-A and analyzed EYFP-CENP-A K124R fusion protein in their RPE-1 CENP-A-/F knockout system. K124 ubiquitin is not expected to bind directly to HJURP based on structural predictions4, however, addition of mono-ubiquitin is predicted to have an impact on protein conformation of CENP-A. The protein of CENP-A conformation can be changed by the presence of a large fusion protein, and this conformational change may mask the structural changes caused by the loss of ubiquitylation. We suggest that the fusion of large-sized protein induces ubiquitylation at a lysine other than K124 in EYFP-CENP-A K124R mutant and this ubiquitylation at another site inhibits/masks the original K124R single mutant phenotype. Evidence that ubiquitylation occurs at different lysine in the CENP-A K124R mutant protein with a large tag protein (EYFP) was reported in our previous publication8. It was found that EYFP tagging induces ubiquitination of another lysine site of EYFP-CENP-A K124R and that EYFP-CENP-A K124R mutant binds to HJURP. As a result, this ubiquitylation at another site inhibits/masks the original K124R single mutant phenotype, and both EYFP-CENP-A WT and K124R mutants showed centromere localization (we used and compared pBabe-EYFP-CENP-A WT and K124R mutant, together with pBabe-EYFP control.). The results demonstrated that Flag-tagged or untagged CENP-A K124R mutants are lethal but can be rescued by a monoubiquitin fusion, suggesting that CENP-A ubiquitylation is indispensable to cell viability.
In recent years, many studies have developed different assays to identify posttranslational modifications (PTMs) of CENP-A protein and other centromere-kinetochore proteins both in vivo and in vitro9,10,11. Analogous to the PTMs of histone proteins that are a major mechanism regulating the function of chromatin, PTMs of centromeric chromatin components are also involved in an essential mechanism to regulate the overall structure and function of centromeres. The majority of CENP-A PTM sites are specific to CENP-A-containing nucleosomes, although a few of them are conserved in histone H3, suggesting that modification of these residues contribute to the centromere-specific function. PTMs of CENP-A including phosphorylation, acetylation, methylation, and ubiquitylation were previously reported9, suggesting that CENP-A is subjected to a variety of PTMs and their combinatorial arrays on its amino terminus and C-terminus histone-fold domain. The importance of CENP-A modifications in multiple functions was revealed by many groups including ours. These functions involve CENP-A deposition at centromeres, protein stability, and recruitment of the CCAN (constitutive centromere-associated network)9. However, limited studies and findings of CENP-A PTMs are preformed where comparisons are made with one of canonical histones that directly or indirectly regulate their function. Technical reports focusing on the methodology to identify these CENP-A PTMs are also limited.
Because CENP-A ubiquitylation is required for CENP-A deposition at the centromere12, inherited through dimerization between cell division5, and indispensable to cell viability8, the method to identify CENP-A ubiquitylation would be essential in future to study the functional activity, positioning, and structure of the centromere. Therefore, here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that the EYFP tagging induces ubiquitylation at a different lysine in the CENP-A K124R mutant protein8. Protocols of other control assays and analyses (immunofluorescence analysis, colony outgrowth assay, and in vivo ubiquitylation assay) are also presented to discuss the outcome of major mass spectrometry analysis properly.

<table>
	<tbody>
		<tr>
			<td>Equpiments/Tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 ml protein low binding tubes</td>
			<td>Eppendorf</td>
			<td>022431064</td>
			<td>For mass spectrometry analysis</td>
		</tr>
		<tr>
			<td>10cm cell culture dish</td>
			<td>BIOFIL/JET, China</td>
			<td>700224</td>
			<td>10 cm tissue culture dish (Yohei lab, PN63)</td>
		</tr>
		<tr>
			<td>6 Well Cell Culture Cluster</td>
			<td>Fisher/Corning Incorporated</td>
			<td>07-200-83</td>
			<td>6-well culture plate</td>
		</tr>
		<tr>
			<td>CentriVap</td>
			<td>LABCONCO</td>
			<td>-</td>
			<td>Benchtop vacuum concentrator for vaccum dry peptides for mass spectrometry analysis</td>
		</tr>
		<tr>
			<td>ChromXP C18CL, 120A, 15 cm x 75 &#956;m</td>
			<td>Eksigent Technologies</td>
			<td>805-00120</td>
			<td>Liquid chromatography (RPLC) column for mass spectrometry analysis</td>
		</tr>
		<tr>
			<td>HCX PL APO 100x oil immersion lens</td>
			<td>Leica</td>
			<td>LEICA HCX PL APO NA 1.40 OIL PHE</td>
			<td>100X Oil immersion lens</td>
		</tr>
		<tr>
			<td>HCX PL APO 63x oil immersion lens</td>
			<td>Leica</td>
			<td>LEICA HCX PL APO NA 1.40 OIL PH 3 CS</td>
			<td>63X Oil immersion lens</td>
		</tr>
		<tr>
			<td>Immobilon-FL PVDF Transfer Membrane</td>
			<td>EMD Millipore</td>
			<td>IPVH00010</td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>Leica DM IRE2 motorized fluorescence microscope</td>
			<td>Leica</td>
			<td>-</td>
			<td>motorized fluorescence microscope</td>
		</tr>
		<tr>
			<td>Leica EL6000 compact light source</td>
			<td>Leica</td>
			<td></td>
			<td>External light source for fluorescent excitation</td>
		</tr>
		<tr>
			<td>Micro Cover glass (22 mm x 22 mm)</td>
			<td>Surgipath</td>
			<td>105</td>
			<td>Cover glass (22 mm x 22 mm)</td>
		</tr>
		<tr>
			<td>Model V16-2 polyacrylamide gel electrophoresis apparatus</td>
			<td>Apogee Electrophoresis/CORE Life Sciences</td>
			<td>31071010</td>
			<td>Gel electrophoresis apparatus I to apply bigger SDS-PAGE gel</td>
		</tr>
		<tr>
			<td>nanoLC.2D</td>
			<td>Eksigent Technologies</td>
			<td>-</td>
			<td>liquid chromatography system for mass spectrometry analysis</td>
		</tr>
		<tr>
			<td>NuPAGE 4%-12% Bis-Tris Protein Gels</td>
			<td>Thermo Fisher</td>
			<td>NP0335BOX</td>
			<td>The commercially available 4%-12% Bis-Tris protein gels for mass spectrometry analysis</td>
		</tr>
		<tr>
			<td>Olympus FLUOVIEW FV3000 confocal laser scanning microscope</td>
			<td>Olympus</td>
			<td>-</td>
			<td>Confocal laser scanning microscope (https://www.olympus-lifescience.com.cn/en/support/ downloads/#!dlOpen=%23detail847250519)</td>
		</tr>
		<tr>
			<td>ORCA-R2 Degital CCD camera</td>
			<td>Hamamatsu</td>
			<td>C10600-10B</td>
			<td>CCD camera</td>
		</tr>
		<tr>
			<td>PAP Pen</td>
			<td>Binding Site</td>
			<td>AD100.1</td>
			<td>For a water repellant barrier in immunofluorescent staining</td>
		</tr>
		<tr>
			<td>TISSUE CULTURE DISHES 10CM</td>
			<td>VWR</td>
			<td>25382-166</td>
			<td>10 cm tissue culture dish</td>
		</tr>
		<tr>
			<td>Vertical electrophoresis for gel running (big size)</td>
			<td>Junyi, China</td>
			<td>JY-SCZ6+</td>
			<td>Gel electrophoresis apparatus II to apply bigger SDS-PAGE gel (Yohei lab, PE23)</td>
		</tr>
		<tr>
			<td>VWR Micro Slides, Frosted</td>
			<td>VWR International</td>
			<td>48312-013</td>
			<td>Micro slides</td>
		</tr>
		<tr>
			<td>Primary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CENP-A antibody</td>
			<td>Stressgen/Enzo Life Sciences</td>
			<td>KAM-CC006</td>
			<td>Mouse monoclonal antibody</td>
		</tr>
		<tr>
			<td>Anti-CENP-B antibody</td>
			<td>Novus Biologicals</td>
			<td>H00001059-B01P</td>
			<td>Mouse monoclonal antibody</td>
		</tr>
		<tr>
			<td>anti-GAPDH</td>
			<td>ABCAM</td>
			<td>ab37168</td>
			<td>Rabbit polyclonal antibody</td>
		</tr>
		<tr>
			<td>anti-GAPDH</td>
			<td>Invitrogen</td>
			<td>PA1987</td>
			<td>Rabbit polyclonal antibody</td>
		</tr>
		<tr>
			<td>anti-GFP antibody</td>
			<td>ANTI #76 (Homemade antibody)</td>
			<td></td>
			<td>Rabbit polyclonal antibody</td>
		</tr>
		<tr>
			<td>anti-HA (3F10)</td>
			<td>Roche</td>
			<td>11815016001</td>
			<td>Rat monoclonal antibody</td>
		</tr>
		<tr>
			<td>anti-HJURP</td>
			<td>Proteintech Group</td>
			<td>15283&#8211;1-AP</td>
			<td>Rabbit polyclonal antibody</td>
		</tr>
		<tr>
			<td>anti-Ubiquitin</td>
			<td>Bethyl Laboratories</td>
			<td>A300-317A-1</td>
			<td>Rabbit polyclonal antibody</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad Protein Assay</td>
			<td>Bio-Rad</td>
			<td>500-0006</td>
			<td>Commercial protein assay reagent I for measurement of protein concentration (compatible with 0.1% SDS)</td>
		</tr>
		<tr>
			<td>Branson SONIFIER 450</td>
			<td></td>
			<td></td>
			<td>Sonicator</td>
		</tr>
		<tr>
			<td>Branson Ultrasonics sonicator Microtip Step, Solid, Threaded 9.5 mm</td>
			<td>VWR Scientific Products Inc.</td>
			<td>33995-325</td>
			<td>Disruptor horn for sonication</td>
		</tr>
		<tr>
			<td>Branson Ultrasonics sonicator Microtip Tapered 6.5 mm</td>
			<td>VWR Scientific Products Inc.</td>
			<td>33996-185</td>
			<td>Microtip for sonication</td>
		</tr>
		<tr>
			<td>Buffer A1</td>
			<td>-</td>
			<td>-</td>
			<td>20 mM Tris-HCl, pH 7.4; 50 mM NaCl; 0.5% Nonidet P-40; 0.5% deoxycholate; 0.5% SDS; 1 mM EDTA; complete EDTA-free protease inhibitor reagent</td>
		</tr>
		<tr>
			<td>Complete EDTA-free protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11-873-580-001</td>
			<td>Complete EDTA-free protease inhibitor reagent for buffer A1</td>
		</tr>
		<tr>
			<td>Coomassie brilliant blue R-250</td>
			<td>BBI Life Sciences</td>
			<td>CAS 6104-59-2</td>
			<td>Coomassie blue solution for mass spectrometry analysis</td>
		</tr>
		<tr>
			<td>Crystal violet solution (2.3% crystal violet, 0.1% ammonium oxylate, 20% ethanol)</td>
			<td>SigmaI-Aldrich</td>
			<td>HT90132-1L</td>
			<td>For colony staining</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>SIGMA-SLDRICH</td>
			<td>D9542</td>
			<td>For nuclear staining</td>
		</tr>
		<tr>
			<td>DMEM: F12 Medium</td>
			<td>ATCC</td>
			<td>30-2006</td>
			<td>DMEM: F12 Medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, certified, heat inactivated, US origin</td>
			<td>Life Technologies/Gibco</td>
			<td>10082</td>
			<td>FBS (fetal bovine serum)</td>
		</tr>
		<tr>
			<td>High-glucose DMEM (Dulbecco&#8217;s modified Eagle&#8217;s medium)</td>
			<td>Life Technologies/BioWhittaker</td>
			<td>12-604</td>
			<td>high-glucose DMEM</td>
		</tr>
		<tr>
			<td>Lipofectamin 3000</td>
			<td>Life Technologies/Invitrogen</td>
			<td>L3000</td>
			<td>Transfection reagent I for chemical transfection</td>
		</tr>
		<tr>
			<td>Lipofectamin 3000, P3000 solution</td>
			<td>Life Technologies/Invitrogen</td>
			<td>L3000</td>
			<td>Transfection reagent II for chemical transfection</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A412-4</td>
			<td>Fixation reagant</td>
		</tr>
		<tr>
			<td>Non fat powdered milk (approved substitution for carnation powdered milk)</td>
			<td>Fisher Scientific</td>
			<td>NC9255871 (Reorder No. 190915; Lot# 90629)</td>
			<td>Non-fat skim milk</td>
		</tr>
		<tr>
			<td>Opti-MEM I</td>
			<td>Life Technologies/Invitrogen</td>
			<td>31985</td>
			<td>Reduced serum media</td>
		</tr>
		<tr>
			<td>p-phenylenediamine</td>
			<td>SIGMA-SLDRICH</td>
			<td>P6001</td>
			<td>For mounting medium</td>
		</tr>
		<tr>
			<td>Penicillin, Streptomycin; Liquid</td>
			<td>Fisher/Gibco</td>
			<td>15-140</td>
			<td>Penicillin-streptomycin</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine SOLUTION</td>
			<td>SIGMA-SLDRICH</td>
			<td>P 8920</td>
			<td>Poly-L-Lysine, 0.1% w/v, in water</td>
		</tr>
		<tr>
			<td>Polyethyleneimine [PEI]; 1.0 mg/ml</td>
			<td>Polysciences</td>
			<td>23966&#8211;2</td>
			<td>Transfection reagent III for chemical transfection</td>
		</tr>
		<tr>
			<td>Protein A sepharose CL-4B beads</td>
			<td>GE Healthcare/Amersham</td>
			<td>17-0963-03</td>
			<td>Protein A sepharose CL-4B beads for in vivo ubiquitylation assays using pQCXIP-EYFP-CENP-A</td>
		</tr>
		<tr>
			<td>Restore Western Blot Stripping Buffer</td>
			<td>Thermo Scientific</td>
			<td>PI21059</td>
			<td>Western Blot Stripping Buffer I</td>
		</tr>
		<tr>
			<td>Sequencing grade trypsin</td>
			<td>Promega</td>
			<td>V5111</td>
			<td>For mass spectrometry analysis</td>
		</tr>
		<tr>
			<td>SuperSignal West Femto Maximum Sensitivity Substrate</td>
			<td>Thermo</td>
			<td>34095</td>
			<td>Ultra-sensitive enhanced chemiluminescent (ECL) substrate</td>
		</tr>
		<tr>
			<td>UltraPure Distilled Water</td>
			<td>Life Technologies/Invitrogen/Gibco</td>
			<td>10977</td>
			<td>Sterile tissue culture grade water</td>
		</tr>
		<tr>
			<td>Western Blot Stripping Buffer II ((50 mM Tris-HCl, pH 6.85; 2% SDS; 50 mM DTT; 100 mM 2-Mercaptoethanol)</td>
			<td>-</td>
			<td>-</td>
			<td>Western Blot Stripping Buffer II</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat Anti-Rabbit IgG</td>
			<td>Life Technologies/Invitrogen</td>
			<td>A11008</td>
			<td>fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Goat Anti-Mouse IgG</td>
			<td>Life Technologies/Invitrogen</td>
			<td>A11005</td>
			<td>fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody)</td>
		</tr>
		<tr>
			<td>Softwares</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acquisition FV31S-SW software</td>
			<td>Olympus</td>
			<td>-</td>
			<td>Sofware C1 (https://www.olympus-lifescience.com.cn/en/support/ downloads/#!dlOpen=%23detail847250519)</td>
		</tr>
		<tr>
			<td>Analysis FV31S-DT software</td>
			<td>Olympus</td>
			<td>-</td>
			<td>Sofware C2 (https://www.olympus-lifescience.com.cn/en/support/ downloads/#!dlOpen=%23detail847250519)</td>
		</tr>
		<tr>
			<td>cellSens Dimension software Ver. 1. 18</td>
			<td>Olympus</td>
			<td>-</td>
			<td>Sofware C3 (https://www.olympus-lifescience.com.cn/en/ software/cellsens/)</td>
		</tr>
		<tr>
			<td>Image Studio Analysis Software Ver 4.0</td>
			<td>LI-COR Biosciences</td>
			<td>-</td>
			<td>Software D</td>
		</tr>
		<tr>
			<td>Molecular Imager Versadoc MP4000 System</td>
			<td>Bio-Rad</td>
			<td>-</td>
			<td>Chemiluminescence imager for immunoblot detection</td>
		</tr>
		<tr>
			<td>Odyssey CLx Infrared imaging System</td>
			<td>LI-COR Biosciences</td>
			<td>-</td>
			<td>Infrared imaging system for immunoblot detection</td>
		</tr>
		<tr>
			<td>OpenCFU saftware</td>
			<td>-</td>
			<td>-</td>
			<td>For colony counting (http://opencfu.sourceforge.net/)</td>
		</tr>
		<tr>
			<td>Openlab version 5.5.2. Scientific Imaging Software</td>
			<td>Improvision/PerkinElmer</td>
			<td>-</td>
			<td>Software A</td>
		</tr>
		<tr>
			<td>ProteinPilot Software version 4.5</td>
			<td>AB SCIEX</td>
			<td>-</td>
			<td>Software F for mass spectrometry analysis</td>
		</tr>
		<tr>
			<td>Quantity One 1-D analysis software</td>
			<td>Bio-Rad</td>
			<td>-</td>
			<td>Software E</td>
		</tr>
		<tr>
			<td>TripleTOF 5600+ System</td>
			<td>AB SCIEX</td>
			<td>-</td>
			<td>Mass spectrometry instrument</td>
		</tr>
		<tr>
			<td>Volocity version 6.3 3D Image Analysis Software (Volocity Acquisition)</td>
			<td>PerkinElmer</td>
			<td>-</td>
			<td>Software B1</td>
		</tr>
		<tr>
			<td>Volocity version 6.3 3D Image Analysis Software (Volocity Quantification)</td>
			<td>PerkinElmer</td>
			<td>-</td>
			<td>Software B2</td>
		</tr>
	</tbody>
</table>

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

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.

EYFP-CENP-A K124 mutant shows ubiquitylation, interaction with HJURP, and no defects in centromere localization neither cell lethality. Here the system reported by Fachinetti et al. (2017)6 was re-constituted: in diploid human (RPE-1) cells carrying one disrupted and one &#8216;&#8216;floxed&#8217;&#8217; CENP-A allele (CENP-A-/F), EYFP-CENP-A was expressed from the pBabe-EYFP retrovirus vector. In this system, the expression of endogenous CENP-A from ...

Watch this Scientific Journal Video about Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A at JoVE.com

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement 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-tagged CENP-A (EYFP-CENP-A) protein.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

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5.5K Views.  Nanjing University. CENP-A ubiquitylation is an important requirement 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-tagged CENP-A (EYFP-CENP-A) protein.

Video: Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...