Name: profiling ubiquitin and ubiquitin-like dependent post-translational modifications and identification of significant alterations
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Description: Watch this Scientific Journal Video about Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations at JoVE.com

This work was supported by La Ligue Contre le Cancer to HV and MS, and the ARC (association pour la recherche sur le cancer) to PS, INCa (institute national du cancer) and Canceropole PACA to JI. The mass spectrometry facility of Marseille Proteomics (marseille-proteomique.univ-amu.fr) supported by IBISA (Infrastructures Biologie Sant&#233; et Agronomie), Plateforme Technologique Aix-Marseille, the Canc&#233;rop&#244;le PACA, the Provence-Alpes-C&#244;te d&#39;Azur R&#233;gion, the Institut Paoli-Calmettes and the Centre de Recherche en Canc&#233;rologie de Marseille.

1. Generation of stable cell lines expressing 6His-Flag-Ubl
NOTE: Co-transfection of HEK-293T cells with pCCL-6HF-Ubl, pVSVG and delta-Helper.
<ol>
	<li>Day 0: Seed 293T cells in a 6-well plate to obtain 50-70% confluence the day after.</li>
	<li>Day 1: Co-transfect 50-70% confluent cells with a mix of 1 &#181;g of pCCL-6HF-Ubl or pCCL-GFP, 1 &#181;g of pVSVG and 1 &#181;g of delta-Helper vectors, using a transfection reagent and protocol for lentivirus production. After 6 h of transfection,...

<ol>
	<li>Prabakaran, S., Lippens, G., Steen, H., Gunawardena, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+modification:+Nature's+escape+from+genetic+imprisonment+and+the+basis+for+dynamic+information+encoding.">Post-translational modification: Nature's escape from genetic imprisonment and the basis for dynamic information encoding.</a> Wiley Interdisciplinary Reviews: Systems Biology and Medicine. , (2012).</li><li>Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+function+of+ubiquitin-like+proteins.">Origin and function of ubiquitin-like proteins.</a> Nature. 458 (7237), 422-429 (2009).</li><li>Kirkpatrick, D. S., Weldon, S. F., Tsaprailis, G., Liebler, D. C., Gandolfi, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+identification+of+ubiquitinated+proteins+from+human+cells+expressing+His-tagged+ubiquitin.">Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin.</a> Proteomics. 5 (8), 2104-2111 (2005).</li><li>Peng, J., 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=A+proteomics+approach+to+understanding+protein+ubiquitination.">A proteomics approach to understanding protein ubiquitination.</a> Nature Biotechnology. 21 (8), 921-926 (2003).</li><li>Matsumoto, 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=Large-scale+analysis+of+the+human+ubiquitin-related+proteome.">Large-scale analysis of the human ubiquitin-related proteome.</a> Proteomics. 5 (16), 4145-4151 (2005).</li><li>Hjerpe, R., Rodr&#237;guez, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+approaches+for+characterizing+ubiquitinated+proteins.">Efficient approaches for characterizing ubiquitinated proteins.</a> Biochemical Society Transactions. 36 (5), 823-827 (2008).</li><li>Bonacci, T., 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=Identification+of+new+mechanisms+of+cellular+response+to+chemotherapy+by+tracking+changes+in+post-translational+modifications+by+ubiquitin+and+ubiquitin-like+proteins.">Identification of new mechanisms of cellular response to chemotherapy by tracking changes in post-translational modifications by ubiquitin and ubiquitin-like proteins.</a> Journal of Proteome Research. 13 (5), 2478-2494 (2014).</li><li>Bedford, L., Lowe, J., Dick, L. R., Mayer, R. J., Brownell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin-like+protein+conjugation+and+the+ubiquitin-proteasome+system+as+drug+targets.">Ubiquitin-like protein conjugation and the ubiquitin-proteasome system as drug targets.</a> Nature Reviews Drug Discovery. 10 (1), 29-46 (2011).</li><li>Hoeller, D., Dikic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+ubiquitin+system+in+cancer+therapy.">Targeting the ubiquitin system in cancer therapy.</a> Nature. 458 (7237), 438-444 (2009).</li><li>Silva, J. C., Gorenstein, M. V., Li, G. Z. Z., Vissers, J. P. C., Geromanos, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+quantification+of+proteins+by+LCMSE:+a+virtue+of+parallel+MS+acquisition.">Absolute quantification of proteins by LCMSE: a virtue of parallel MS acquisition.</a> Molecular &amp; Cellular Proteomics. , (2006).</li><li>Kim, W., 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=Systematic+and+quantitative+assessment+of+the+ubiquitin-modified+proteome.">Systematic and quantitative assessment of the ubiquitin-modified proteome.</a> Molecular Cell. 44 (2), 325-340 (2011).</li></ol>

We have developed a robust and reliable methodology to generate profiles of proteins modified by the main ubiquitin family members. Indeed, we have successfully applied this protocol to generate profiles of PTMs by ubiquitin, and also by SUMO and Nedd8, and to detect alterations associated with a treatment7, in response to the over expression or knockdown of a certain gene (data not shown) and in cells that acquired a resistant phenotype to diverse chemotherapeutic drugs.
<p class="jove_conten...

The method proposed here is dedicated to study PTMs mediated by the ubiquitin family members from cultured mammalian cells in order to identify potential alterations associated with a specific condition (treatment, differentiation, etc). PTMs represent the last step of regulation of proteins&#39; functions1. Indeed, once produced by the translational machinery, most if not all proteins undergo different kinds of PTMs that modulate their activity, molecular interactions, and intracellular location1. Among the plethora of PTMs are the ones mediated by the ubiquitin family of proteins, ubiquitin itself and all ubiquitin-likes, have the potential to regulate all intracellular or partially cytoplasmic proteins2. Because they are themselves proteins, they can be conjugated to each other, forming homogeneous and heterogeneous chains of diverse topologies, each associated with specific regulatory functions2. Tools are needed to try to decipher and understand this complex machinery. Many approaches were developed worldwide, having their own advantages and disadvantages, and here we propose one with high performance suitable for cultured cells.
The main advantage of this method is its accuracy. Indeed, the purity of isolated modified proteins is highly improved by the combinatorial use of the two tags (6His and Flag) and the two step-procedure and therefore it is much more selective than a single tag fusion Ub/Ubl3,4. The presence of the 6His tag enables a first step of purification in a fully denaturing condition thereby avoiding any co-purification of proteins containing ubiquitin binding domains or other proteins binding to the ubiquitinated ones. This is a technical problem encountered by several other approaches based on affinity purification of ubiquitinated proteomes using either specific antibodies5 or tandem ubiquitin binding elements (TUBEs)6. Importantly, this technique is not biased in favor of purification of a certain type of ubiquitination, as it could be the case for some other approaches, since both mono and different kinds of polyubiquitinations were identified7. Consequently, once found, an alteration of ubiquitination will have to be studied in more details by standard biochemical approaches in order to identify the exact kind of ubiquitination involved.
Finally, another technical advantage of this protocol is the use of lentiviruses, that easily and rapidly creates stable expressing cell lines with reasonable level of expressions of tagged modifier without interfering with the normal cellular behavior.
Whereas one important role of ubiquitination is to target proteins for proteasomal degradation, it is now known that it has many other regulatory properties for potentially most intracellular or partially intracellular proteins1. The number of these functions is further augmented by the existence of many ubiquitin like proteins, forming a family of proteins regulating almost every cell mechanisms1. Their alterations can have drastic impact on the cell biology and can lead or participate in pathological situations8, such as cancer9. Hence, tools are needed to explore this vast landscape and identify the alterations associated with a pathological condition that could serve as novel therapeutic targets.
This protocol is dedicated to cells in culture since they need to be transduced to express exogenous tagged Ub/Ubl. Once created, these stable cell lines can be used to generate Ubl profiles from culture in 2D or 3D or xenografts, thereby extending the horizon of the different experimental models that can be applied to study PTMs profiles.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ANTI-FLAG M2 Affinity Gel</td>
			<td>Sigma-Aldrich</td>
			<td>A2220-5ML</td>
			<td>binds all Flag tagged proteins</td>
		</tr>
		<tr>
			<td>anti-Flag M2 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>F3165</td>
			<td>to detect 6His-Flag tagged expression of ub/ubl</td>
		</tr>
		<tr>
			<td>Cell strainer 40 &#181;m</td>
			<td>Falcon</td>
			<td>352350</td>
			<td>to remove floating pellet from guanidine lysed cells</td>
		</tr>
		<tr>
			<td>Flag peptide</td>
			<td>Sigma-Aldrich</td>
			<td>F3290</td>
			<td>elute flag tagged proteins from anti-flag beads</td>
		</tr>
		<tr>
			<td>Guanidine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>50933</td>
			<td>chaotropic agent used to denature all proteins in cell lysate</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I5513</td>
			<td>eluates 6His bond proteins from Ni-NTA beads</td>
		</tr>
		<tr>
			<td>Lipofectamine 3000</td>
			<td>ThermoFisher</td>
			<td>L3000015</td>
			<td>to transfect HEK-293T cells to produce lentiviruses</td>
		</tr>
		<tr>
			<td>Lobind tubes</td>
			<td>Sigma-Aldrich</td>
			<td>Z666491</td>
			<td>avoids absorption of precious material</td>
		</tr>
		<tr>
			<td>Membrane Filter, 0.45 &#181;m</td>
			<td>Millipore</td>
			<td>HAWP04700F1</td>
			<td>to filter the lentiviral supernantant</td>
		</tr>
		<tr>
			<td>Ni-NTA</td>
			<td>Qiagen</td>
			<td>30210</td>
			<td>purification of the 6His tag</td>
		</tr>
	</tbody>
</table>

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.

Transduction of culture mammalian cells to create GFP and 6HF-Ub expressing cells 
To produce lentiviruses which will be used later to transduce MiaPaCa-2 cells, 70% confluent HEK-293T cells are co-transfected with an equal amount of the three vectors, pCCL-6HF-Ubiquitin or GFP/Delta-Helper/pvSvG. After 24 h of production, the medium containing lentiviral particles is recovered and filtered. It is possible at this point to control the efficiency of the transfection b...

Watch this Scientific Journal Video about Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations at JoVE.com

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

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.

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

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