Name: mass spectrometry analysis to identify ubiquitylation of eyfp-tagged cenp-a (eyfp-cenp-a)
Uploaded: 2020-06-10T13:00:00.000+00:00
Duration: 9 min 2 s
Description: Watch this Scientific Journal Video about Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A at JoVE.com

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.

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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>&lt;strong&gt;Equpiments/Tools&lt;/strong&gt;</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 &amp;mu;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>&lt;strong&gt;Primary antibodies&lt;/strong&gt;</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&amp;ndash;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>&lt;strong&gt;Reagents&lt;/strong&gt;</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&amp;rsquo;s modified Eagle&amp;rsquo;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&amp;ndash;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>&lt;strong&gt;Secondary antibodies&lt;/strong&gt;</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>&lt;strong&gt;Softwares&lt;/strong&gt;</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 조회수.  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.

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

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle 1,3,6,7.
The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate 8-13. However, the polyacrylamide-gel-based techniques, which used the "native" proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs.
Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) 9,10,14,15. The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate.
We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate 16. We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat/KM was obtained as (3.2 &plusmn; 0.55) x107 M-1s-1 of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.

Quantitative FRET F&#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

A novel method involving quantitative analysis of FRET (F&ouml;rster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.

정량은 SENP1 단백 분해 효소 반응 속도론 결정에 대해 (포스터 공명 에너지 전송) 분석 안달

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

연구

생체공학

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

 종속 키 1의 시험관에서 키에 의해 인 산화 특정 사이트 분석 결과

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

생화학

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

펩타이드 농축 및 질량 분광법에 의한 단백질 유비퀴틴화 부위 검출

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

유비퀴틴 및 유비퀴틴과 같은 종속 적 번역 후 수정 및 중대한 변경 식별 프로파일링

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

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.

This method describes the assessment of global changes in ubiquitin chain topology. The assessment is performed by the application of a mass spectrometry-based targeted proteomics approach.

병렬 반응 모니터링에 의한 유비퀴틴 연쇄 분석

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The ...

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, including cell survival, proliferation, and differentiation. It is a reversible process that consists of a covalent attachment of ubiquitin to the substrate through a cascade reaction of at least three different enzymes, composed of E1 (Ubiquitin-activation enzyme), E2 (Ubiquitin-conjugating enzyme), and E3 (Ubiquitin-ligase enzyme). The E3 complex plays an important role in substrate recognition and ubiquitylation. Here, a protocol is described to evaluate substrate ubiquitylation in mammalian cells using transient co-transfection of a plasmid encoding the selected substrate, an E3 ubiquitin ligase, and a tagged ubiquitin. Before lysis, the transfected cells are treated with the proteasome inhibitor MG132 (carbobenzoxy-leu-leu-leucinal) to avoid substrate proteasomal degradation. Furthermore, the cell extract is submitted to small-scale immunoprecipitation (IP) to purify the polyubiquitylated substrate for subsequent detection by western blotting (WB) using specific antibodies for ubiquitin tag. Hence, a consistent and uncomplicated protocol for ubiquitylation assay in mammalian cells is described to assist scientists in addressing ubiquitylation of specific substrates and E3 ubiquitin ligases.

We provide a detailed protocol for a ubiquitylation assay of a specific substrate and an E3 ubiquitin-ligase in mammalian cells. HEK293T cell lines were used for protein overexpression, the polyubiquitylated substrate was purified from cell lysates by immunoprecipitation, and resolved in SDS-PAGE. Immunoblotting was used to visualize this post-translational modification.

포유동물 세포 용해물에서 E3 유비퀴틴-리가아제에 의한 기질 유비퀴틸화의 평가

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, ...

Ubiquitylated, SUMOylated 및 ADP-Ribosylated DNA-Protein Crosslinks 측정

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

저자 스포트라이트: DNA 단백질 교차결합의 정량적 검출 및 번역 후 변형(Post-Translational Modifications)  

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-단백질 가교 및 번역 후 변형의 정량적 검출

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

의 단백질 단지의 식별<em&gt; 대장균</em&gt; 직렬 질량 분석법과 함께 순차적 펩타이드의 동질 정화를 사용하여

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

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

요약

더 많은 비디오 탐색

이 비디오의 챕터

정량은 SENP1 단백 분해 효소 반응 속도론 결정에 대해 (포스터 공명 에너지 전송) 분석 안달

 종속 키 1의 시험관에서 키에 의해 인 산화 특정 사이트 분석 결과

펩타이드 농축 및 질량 분광법에 의한 단백질 유비퀴틴화 부위 검출

유비퀴틴 및 유비퀴틴과 같은 종속 적 번역 후 수정 및 중대한 변경 식별 프로파일링

병렬 반응 모니터링에 의한 유비퀴틴 연쇄 분석

포유동물 세포 용해물에서 E3 유비퀴틴-리가아제에 의한 기질 유비퀴틸화의 평가

DNA-단백질 가교 및 번역 후 변형의 정량적 검출

DNA-단백질 가교 및 번역 후 변형의 정량적 검출

DNA-단백질 가교 및 번역 후 변형의 정량적 검출

의 단백질 단지의 식별