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, change the medium to a fresh one corresponding to cells to be transduced. Seed the cells to be transduced in a 6 well plate in order to obtain a 10-20% confluence the day after (the day of starting the transduction).</li>
	<li>Day 2: 24 h after transfection, recover the medium containing lentiviral particles and filter using 0.45 &#181;m filters. If needed, add fresh medium at this point in order to produce a second batch of lentiviruses. Replace the medium of cells to be transduced (10-20% confluence) by the one containing lentiviruses. 
	NOTE: Lentiviral medium can be kept at +4 &#176;C for several days before transduction or stored at -80 &#176;C for months.</li>
	<li>Incubate the cells with lentiviruses between 24 h to 72 h in a standard incubator (37 &#176;C, 5% CO2), and then change the medium for fresh standard one. If possible, check GFP expression using an inverted fluorescent microscope to evaluate efficiency of transduction: percentage of expressing cells and relative level of expression per cell. If no fluorescence is detected, wait for additional 2-3 days as expression may take longer depending on cells type to be transduced.</li>
	<li>If GFP control is positive, grow all cells until having enough to perform an expression control of 6HF-Ubl by immunofluorescence and Western blot using anti-Flag antibody.</li>
</ol>
2. Double purification of modified proteins
NOTE: Buffer 1: 6 M Guanidinium-HCl, 0.1 M Na2HPO4/NaH2PO4, pH 8.0, 0.5% Triton X-100. 
Buffer 2: 50 mM NaH2PO4, 150 mM NaCl, 1% Tween20, 5% Glycerol, pH 8.0. 
Buffer 3: 100 mM NH4HCO3, pH 8.0.
<ol>
	<li>Cell lysis: Once ready, wash culture dishes at least one time with phosphate-buffered saline (PBS) at room temperature (RT) and proceed to cell lysis or, alternatively, flash freeze in liquid N2 and store at -80 &#176;C. For lysis, add 2 mL of Buffer 1 per a 15 cm dish at RT. Use a cell scraper to recover all lysates in 50 mL conical centrifuge tubes (final volume about 20 mL).</li>
	<li>Sonicate the lysates three times for 30 s separated by a 1 min pause.</li>
	<li>Centrifuge the sonicated lysates at 15,000 x g for 15 min.</li>
	<li>Transfer the supernatant to a new tube using a cell strainer (40 &#181;m).</li>
	<li>Determine samples&#39; concentration and adjust, if necessary, to obtain the same amount of proteins and same volume. Use a total amount of protein between 50 and 100 mg (10 dishes with 15 cm diameter for MiaPaCa-2 cells).</li>
	<li>Add Ni2+-NTA beads, using 2 &#181;L of beads per 1 mg of protein.</li>
	<li>Rotate at 30 rpm during 2.5 h at RT.</li>
	<li>Pellet the beads at 500 x g for 5 min.</li>
	<li>Wash the beads with 1 mL of Buffer 1, transferring the samples to a 1.5 mL microcentrifuge tube, then transfer the tubes on ice. Perform all the next steps on ice or at 4 &#176;C.</li>
	<li>Wash two times with 1 mL of ice-cold Buffer 2 containing 10 mM imidazole.</li>
	<li>To elute bound proteins, add 600 &#181;L of Buffer 2 containing 250 mM imidazole and rotate for 2 h at 4 &#176;C.</li>
	<li>Pellet the beads by centrifugation at 500 x g for 1 min. Transfer the supernatants to new, pre-cooled, 1.5 mL tubes and add 50 &#181;L of anti-Flag M2 antibody conjugated beads.</li>
	<li>Rotate at 30 rpm for 2.5 h at 4 &#176;C, then wash 2 times with 500 &#181;L of Buffer 2, then 2 times with 500 &#181;L of Buffer 3.</li>
	<li>For the final elution, add 100 &#181;L of Buffer 3 containing a Flag peptide at 0.1 &#181;g/&#181;L and rotate at 4 &#176;C for 1.5 h.</li>
	<li>Centrifuge at 500 x g for 1 min and transfer the supernatants to new pre-cooled tubes.</li>
	<li>Take 10% (10 &#181;L) to load on SDS-PAGE and perform a silver staining of the gel to control the purification quality. If the purification looks good, analyze the 90% left by LC-MS/MS.</li>
</ol>
3. Processing of mass spectrometry data to generate profiles of Ub/Ubls PTMs and to identify significant differences between them
NOTE: Results from MS analysis contain many information including the total number of peptides as well as the peak area values (mean of TOP 3 peptide area10) for each protein identified in each samples. These data can be processed using either the peptide count numbers or the peak area values, or both. For calculation with peak areas, because these values are usually in the range of 106, it is necessary to divide them by this order before applying the same formulas as below. The results obtained with both methodologies of counting should show a strong correlation as it usually does. For each identified protein, use the following formulas where: 
v1 <img src="/files/ftp_upload/60402/60402eq1.jpg" /> peptides values in non-treated ubiquitin sample (e.g., Ub - drug) 
v2 <img src="/files/ftp_upload/60402/60402eq1.jpg" /> peptides values in Gemcitabine treated ubiquitin sample (e.g., Ub + drug) 
k1 <img src="/files/ftp_upload/60402/60402eq1.jpg" /> peptides values in non-treated control GFP sample (e.g., GFP - drug) 
k2 <img src="/files/ftp_upload/60402/60402eq1.jpg" /> peptides values in Gemcitabine treated control GFP sample (e.g., GFP + drug).
<ol>
	<li>Normalization: Normalize values between drug treated cells and untreated cells for Ubiquitin and GFP using the following formulas. Normalized v = V and normalized k = K. 
	V1=v1.(&#8721;v1+ &#8721;v2) / (2. &#8721;v1) ; V2=v2.(&#8721;v1+ &#8721;v2) / (2. &#8721;v2) 
	K1=k1.(&#8721;k1+ &#8721;k2) / (2. &#8721;k1) ; K2=k2.(&#8721;k1+ &#8721;k2) / (2. &#8721;k2)</li>
	<li>Removal of background: Using the following formulas, subtract values in control sample (GFP) from values in the ubiquitin sample to obtain specific values (V&#39;1 and V&#39;2) for each identified protein in both conditions. 
	V&#39;1=V1-K1 if V1-K1&#8805;0 ; V&#39;1=0 if V1-K1&#60;0 
	V&#39;2=V2-K2 if V2-K2&#8805;0 ; V&#39;2 =0 if V2-K2&#60;0</li>
	<li>Variation (Var) of ubiquitination. To obtain a score (between -100 and +100) for positive and negative variations of PTMs induced by a drug, use the following formula in which the difference between specific values of treated and untreated samples are divided by the sum of all values, including those in control (to penalize proteins also identified in control GFP), and multiply by 100. 
	Var = (V&#39;2-V&#39;1)/(V1+K1+V2+K2)*100 ; -100&#60;Var&#60;100 ; 
	Variations below -50 (repression of PTM) or above 50 (induction of PTM) are usually considered as significant.</li>
	<li>Confidence (Conf). Use the following formula to obtain a confidence value between 0 and 100%,: 
	Conf = ((V1+V2)2/(1+V1+V2+K1+K2)2)*100 - 100/(1+V&#39;1+V&#39;2) ; =0 if &#60;0 
	Values above 50 are usually considered to be confident.</li>
	<li>To obtain a nicer distribution of induction/repression values and to consider both variation and confidence parameters, multiply Var and Conf values using the following formula where V <img src="/files/ftp_upload/60402/60402eq1.jpg" /> Var and C <img src="/files/ftp_upload/60402/60402eq1.jpg" /> Conf 
	=SI(V2&#62;0;((V2*C2)^2)/(10^6);-((V2*C2)^2)/(10^6)) 
	NOTE: As peak area values are usually more accurate than peptide counting, it is possible to use specific software which are dedicated to the interpretation of this kind of data such as Perseus (https://www.biochem.mpg.de/5111810/perseus), following recommendations of use.</li>
</ol>

<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>

The authors have nothing to disclose.

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.
There are only few critical steps during the procedure that the manipulator should be careful with. Upon pipetting of beads (Ni-NTA or anti-Flag coupled), it is important to resuspend them thoroughly, especially the anti-Flag beads since are suspended in a viscous glycerol based buffer, and to cut a bit the end of the tip to increase the section. Another precaution should be followed is to pipette slowly in order to avoid stacking of beads. Additional critical steps are the elution with imidazole and then with Flag peptide. A clean Hamilton syringe must be used to recover the supernatant after elution to avoid, as much as possible pipetting of the beads on which nonspecific proteins remain.
Depending on the cell type and the amount of required final material for mass spectrometry, the starting material may be increased or decreased accordingly. Then, the only important adjustment resides in the volume of nickel beads as it should be of 2 &#181;L per 1 mg of proteins in lysate. It may happen that the amount of purified proteins is too low. The expression level of tagged Ub/Ubl should be controlled and, if it is too low, cells can be re-transduced using the same lentiviruses. Alternatively, it is possible to increase the amount of starting material. Sometimes, the amount of nonspecific purified material in GFP or parental cells is too high. One solution to overcome this problem is increasing the volume and the number of washes at both Ni-NTA and Flag purification steps. It is also possible to increase the concentration of imidazole in washes with guanidine buffer, up to 20 mM. Inversely, it is not necessary to increase the concentration of Flag peptide in the final elution step as it will not increase the elution. It is more important to use fresh peptide as over time, even if stored at -20 &#176;C, the efficacy of the peptide may decrease.
The main limitation of this protocol is that it is only suitable for cultured cells because they have to be transduced to express the desired tagged Ubl. Hence, any study on tissues or tumor samples has to be performed using alternative approaches such as diGly peptides enrichments after trypsin digestion11, immunoprecipitation with antibodies specific to the Ub/Ubl of interest5, or use of TUBEs6. All these alternative methods are also suitable for their application in cultured cells. They have the advantage of using the endogenous Ub/Ubls machinery. However, several drawbacks exist. Immunoprecipitations are difficult to be performed with a low background and, like TUBEs approaches, proteins interacting with modified proteins, and even the modifier itself, cannot be eliminated, even though practical improvements have been obtained using more stringent conditions. DiGly peptides enrichment is a very powerful technic but may correspond to different kind of PTMs. For example, diGly enrichment from a trypsin digested lysate will identify mainly ubiquitinated proteins but also neddylated ones, as Nedd8 leaves the same remnant diGly signature as ubiquitin does11.
Another limitation of this protocol is that the presence of the 6His-Flag tag may alter the normal function of Ub/Ubl and the overexpression by itself could alter the machinery. This could for example impede the generation of polyubiquitin chains and favor monoubiquitination. However, since both mono multi and polyubiquitin chains have been identified using this protocol, it seems that it is not the case7. The presence of the tag at the N-terminus also prevents the formation of linear ubiquitination, where ubiquitin moieties are linked together via the N-terminal methionine of ubiquitin. However, even though 6HF-udiquitin cannot be ubiquitinated on its first Met residue, it still has the ability to end this kind of chain.
Also, whereas it is a substantial advantage to use lentiviruses which enables the creation of stable cells lines in one or two weeks, it is possible that not all cells would express the desired construct. This may be not a real problem for the purification procedure as long as enough cells express the exogenous Ub/ubl, but the problem may come with long term culture as over several passages there could be a clonal variation with enrichment in cells with less or no expression. This drawback, however, can be easily bypassed by using lentiviruses containing either a resistance selection gene or a fluorescent protein which can be use in flow cytometry to select only positive cells.

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 by checking the green fluorescence of GFP expressing 293T cells on an inverted microscope. It should be near 100% of cells. MiaPaCa-2 cells are incubated with lentiviral supernatant for 1 to 3 days. The efficacy of lentiviral transduction is first controlled by looking at the GFP fluorescence of GFP transduced cells (Figure 1A upper panel). The GFP expression level may vary from one cell to another but 100% of them should be fluorescent. Once this control is done, the expression of Flag-ubiquitin will have to be also controlled. This is done by immunofluorescence staining using an anti-Flag antibody (usually M2 monoclonal) (Figure 1A lower panel). This will show the percentage of transduced cells, which should be 100% to guarantee a stable expression over future passages of cell culture. In order to control the expression level of exogenous Flag-ubiquitin, lysates from transduced cells are analyzed by SDS-PAGE followed by Western blot with anti-Flag antibody (Figure 1B). Both cell lines can be frozen.
Two steps purification and control by SDS PAGE and silver staining 
Once the stable expressing cell lines have been validated, both GFP and 6HF-ubiquitin cells are amplified until enough material is obtained in order to proceed with the two-step purification. It is recommended to keep a backup of these cell lines as frozen stock in liquid nitrogen in order to be able to thaw them when needed. 36 h before processing, half of the cells (half GFP and half 6HF-Ubiquitin) are treated with 10 &#181;M of Gemcitabine. When ready, ubiquitinated proteins are purified from 6HF-ubiquitin and from GFP control cells, using the two-step purification protocol (Figure 2A). 10% of the final elution is used to control the amount and integrity of purified material by SDS-PAGE and silver staining of the gel (Figure 2B). Molecular weight markers bands can be used to estimate the amount of purified ubiquitinated proteins. Alternatively, a known amount of BSA or any other protein can be loaded in a line of the gel to help quantifying the purified proteins. Once this verification is done, the remaining 90% of samples are analyzed by liquid chromatography coupled to tandem mass spectrometry to allow identification and semi-quantitation of purified proteins.
Identification of ubiquitinated proteins by background (GFP samples) subtraction 
Data from LC-MS/MS analysis of the samples give the names and quantifications (peak areas and number of observed peptides) of each identified protein in GFP and 6HF-Ub samples. The proteins having the highest quantification in ubiquitin sample compared to GFP sample are most likely to be the ones really ubiquitinated and applying the formulas described in methods above allows their classification by giving a confidence score from 0 to 100%. Proteins identified with a score above 50% are considered ubiquitinated ones (Figure 3A).
This step which basically removes background proteins (GFP) from ubiquitin samples led to the identification of 364 proteins significantly ubiquitinated (Figure 3B)7. Note here that the proteins identified with the highest scores are mostly already known as main targets of ubiquitination which proves the efficacy of this purification method.
Then it is possible to perform a gene set enrichment analysis (GSEA) of these ubiquitinated proteins in order to highlight the biological processes in which they are involved (Figure 3C), their molecular functions, their cellular compartment, or any other gene ontology categorization. It is interesting to compare this ubiquitinated proteome with the full proteome of the cell when possible. Indeed, this analysis reveals the real contribution of these ubiquitinated proteins in specific processes such as translation or proteolysis for example (Figure 3C).
The main purpose of this kind of experiment is to identify alterations within the ubiquitinated proteome induced by a treatment for example, here gemcitabine. By comparing the ubiquitinome (the ubiquitinated specific proteome) in treated and untreated cells using the specific formula yields a value from -100 (repression of ubiquitination) to +100 (induction of ubiquitination). Considering only the values below -50 or above +50 as significant, a total of 73 induced ubiquitinations and 29 repressed ubiquitinations have been identified (Figure 4A). GSEA analysis of these alterations of ubiquitination revealed specific enrichments in DNA repair processes or cell cycle, and important variations in translation and RNA metabolic processes (Figure 4B). This result is highly logical since gemcitabine is a base analogue that blocks DNA synthesis and provokes DNA damages.
To go further, it is also interesting to use databases of interacting proteins in order to explore and validate potential interacting networks formed by gemcitabine induced alterations of ubiquitination (Figure 5). This led to the identification of functional interacting networks strongly affected by increased or decreased ubiquitination of the involved proteins.
Finally, among the altered ubiquitination, some may involve proteins of highest interest, due to their known or potential functions according to literature. Hence, one important step is to validate that what is observed by mass spectrometry is real and trustworthy. PCNA was one of the most over-ubiquitinated protein upon gemcitabine treatment detected by mass spectrometry analysis (Figure 4A). To verify that gemcitabine indeed induces the ubiquitination of PCNA, MiaPaCa-2 cells expressing 6HF-Ub and GFP are grown, treated or not, and subject to either Ni-NTA purification in denaturing conditions or to anti-Flag immunoprecipitation followed by Flag peptide elution in native conditions, resolved on SDS PAGE followed by western blot with the corresponding antibody. The result shown in Figure 6 confirmed that indeed PCNA is strongly ubiquitinated in response to gemcitabine treatment in MiaPaCa-2 cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60402/60402fig1.jpg" /> 
Figure 1: Establishment of stable cell lines expressing 6His-Flag-Ubl. (A) Control of GFP expression in GFP transduced cells (upper panel) and of 6His-Flag-Ubiquitin by immunofluorescence using anti-Flag (M2) antibody as primary and alexa-567 anti-mouse secondary antibody. DAPI was used to stain nuclei. Scale bar: 50 &#181;m. (B) Control of 6His-Flag-Ubiquitin expression in cell lysates by Western blot using anti-Flag antibody (Alternatively, an anti-6his antibody can be used) on the left, and anti-ubiquitin antibody, on the right, to compare the expression of 6His-Flag-Ubiquitin (6HF-Ubiq) with endogenous Ubiquitin (Endo. Ubiq). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60402/60402fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60402/60402fig2.jpg" /> 
Figure 2: Two step purification of ubiquitinated proteins. (A) Schematic representation of the procedure. (B) 10% of the final elution was subjected to SDS PAGE followed by silver staining of the gel in order to estimate the quantity and purity (compared to GFP) of isolated proteins. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60402/60402fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60402/60402fig3.jpg" /> 
Figure 3: Identification of ubiquitinated proteins (adapted from Bonacci et al.7). (A) Relative amount of specific (ubiquitin sample) and non-specific (GFP sample) peptides for each identified protein were plotted in function of their confident scores. As shown, the proportion of non-specific over ubiquitinated proteins becomes too important below the score of 50. Hence, only proteins identified with a score superior to 50 are considered significant. (B) Table showing the 20 best ubiquitinated proteins among the 364-total identified. (C) Repartition of ubiquitinated proteins and total proteins of MiaPaCa-2 cells within biological processes (Values &#62; 1.5% were considered only). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60402/60402fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60402/60402fig4.jpg" /> 
Figure 4: Gemcitabine induced alterations of PTM profiles (adapted from Bonacci et al.7). (A) Listing of the 20 proteins with highest increased (total 73) or decreased (total 29) ubiquitination upon gemcitabine treatment (Conf: confidence; Ind: induction; Rep: repression). (B) Repartition of Gemcitabine induced altered ubiquitination within biological processes and comparison with non-treated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60402/60402fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60402/60402fig5.jpg" /> 
Figure 5: Functional interactomes of gemcitabine induced altered ubiquitination. Potential interactions between all proteins with gemcitabine induced alteration of ubiquitination are identified using a protein-protein interactions database (STRING: string-db.org). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60402/60402fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60402/60402fig6.jpg" /> 
Figure 6: Biochemical validations of interesting gemcitabine induced alterations of ubiquitination. In order to validate the increased ubiquitination of PCNA after gemcitabine treatment, lysates of cells expressing 6HF-Ubiquitin, treated or not with gemcitabine, were subjected to Nickel pull-down (Ni-NTA) followed by anti-PCNA Western blot. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60402/60402fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

profiling-ubiquitin-ubiquitin-like-dependent-post-translational

Nanyang Technological University

Research

JoVE Journal

Biochemistry

5.5K Views.  Institut Paoli-Calmettes, CRCM. 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.

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

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which may be essential for a given protein&#39;s role both physiologically and pathologically. Enrichment of PTMs is often necessary when investigating the PTM status of a target protein, because PTMs are often transient and relatively low in abundance. Many pitfalls are encountered when enriching for a PTM of a target protein, such as buffer incompatibility, the target protein antibody is not IP-compatible, loss of PTM signal, and others. The degree of difficulty is magnified when investigating multiple PTMs like acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation for a given target protein. Studying a combination of these PTMs may be necessary, as crosstalk between PTMs is prevalent and critical for protein regulation. Often, these PTMs are studied in different lysis buffers and with unique inhibitor compositions. To simplify the process, a unique denaturing lysis system was developed that effectively isolates and preserves these four PTMs; thus, enabling investigation of potential crosstalk in a single lysis system. A unique filter system was engineered to remove contaminating genomic DNA from the lysate, which is a problematic by-product of denaturing buffers. Robust affinity matrices targeting each of the four PTMs were developed in concert with the buffer system to maximize the enrichment and detection of the endogenous states of these four PTMs. This comprehensive PTM detection toolset streamlines the process of obtaining critical information about whether a protein is modified by one or more of these PTMs.

Investigating multiple, endogenous post-translational modifications for a target protein can be extremely challenging. Techniques described here utilize an optimized lysis buffer and filter system developed with specific PTM-targeting, affinity matrices to detect the acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation modifications for a target protein using a single, streamlined system.

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which ...

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

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.

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; ...

Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods

Immunohistochemistry (IHC) is a highly sensitive and specific technique involved in the detection of target antigens in tissue sections with labeled antibodies. It is a multistep process in which the optimization of each step is crucial to obtain the optimum specific signal. Through IHC, the distribution and localization of specific biomarkers can be detected, revealing information on evolutionary conservation. Moreover, IHC allows for the understanding of expression and distribution changes of biomarkers in pathological conditions, such as obesity. IHC, mainly the immunofluorescence technique, can be used in adult zebrafish to detect the organization and distribution of phylogenetically conserved molecules, but a standard IHC protocol is not estasblished. Orexin and endocannabinoid are two highly conserved systems involved in the control of food intake and obesity pathology. Reported here are protocols used to obtain information about orexin peptide (OXA), orexin receptor (OX-2R), and cannabinoid receptor (CB1R) localization and distribution in the gut and brain of normal and diet-induced obese (DIO) adult zebrafish models. Also described are methods for immunoperoxidase and double immunofluorescence, as well as preparation of reagents, fixation, paraffin-embedding, and cryoprotection of zebrafish tissue and preparation for an endogenous activity-blocking step and background counterstaining. The complete set of parameters is obtained from previous IHC experiments, through which we have shown how immunofluorescence can help with the understanding of OXs, OX-2R, and CB1R distribution, localization, and conservation of expression in adult zebrafish tissues. The resulting images with highly specific signal intensity led to the confirmation that zebrafish are suitable animal models for immunohistochemical studies of distribution, localization, and evolutionary conservation of specific biomarkers in physiological and pathological conditions. The protocols presented here are recommended for IHC experiments in adult zebrafish.

Presented here are protocols for immunohistochemical characterization and localization of orexin peptide, orexin receptors, and endocannabinoid receptors in the gut and brains of normal and diet-induced obesity (DIO) adult zebrafish models using immunoperixidase and double immunofluorescence methods.

Immunohistochemistry (IHC) is a highly sensitive and specific technique involved in the detection of target antigens in tissue sections with labeled ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent attachment of 76 amino acid ubiquitin modifiers to a target protein, depending on the length and topology of the polyubiquitin chain, can result in different outcomes ranging from protein degradation to changes in the localization and/or activity of modified protein. Three enzymes sequentially catalyze the ubiquitination process: E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. E3 ubiquitin ligase determines substrate specificity and, therefore, represents a very interesting study subject. Here we present a comprehensive approach to study the relationship between the enzymatic activity and function of the RING-type E3 ubiquitin ligase. This four-step protocol describes 1) how to generate an E3 ligase deficient mutant through site-directed mutagenesis targeted at the conserved RING domain; 2&#8211;3) how to examine the ubiquitination activity both in vitro and in planta; 4) how to link those biochemical analysis to the biological significance of the tested protein. Generation of an E3 ligase-deficient mutant that still interacts with its substrate but no longer ubiquitinates it for degradation facilitates the testing of enzyme-substrate interactions in vivo. Furthermore, the mutation in the conserved RING domain often confers a dominant negative phenotype that can be utilized in functional knockout studies as an alternative approach to an RNA-interference approach. Our methods were optimized to investigate the biological role of the plant parasitic nematode effector RHA1B, which hijacks the host ubiquitination system in plant cells to promote parasitism. With slight modification of the in vivo expression system, this protocol can be applied to the analysis of any RING-type E3 ligase regardless of its origins.

The goal of this manuscript is to present an outline for the comprehensive biochemical and functional studies of the RING-type E3 ubiquitin ligases. This multistep pipeline, with detailed protocols, validates an enzymatic activity of the tested protein and demonstrates how to link the activity to function.

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent ...

Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They allow the analysis of the intrinsic properties of microtubules, such as dynamics, and their interactions with microtubule-associated proteins (MAPs). The &#8220;tubulin code&#8221; is an emerging concept that points to different tubulin isotypes and various posttranslational modifications (PTMs) as regulators of microtubule properties and functions. To explore the molecular mechanisms of the tubulin code, it is crucial to perform in vitro reconstitution experiments using purified tubulin with specific isotypes and PTMs.
To date, this was technically challenging as brain tubulin, which is widely used in in vitro experiments, harbors many PTMs and has a defined isotype composition. Hence, we developed this protocol to purify tubulin from different sources and with different isotype compositions and controlled PTMs, using the classical approach of polymerization and depolymerization cycles. Compared to existing methods based on affinity purification, this approach yields pure, polymerization-competent tubulin, as tubulin resistant to polymerization or depolymerization is discarded during the successive purification steps.
We describe the purification of tubulin from cell lines, grown either in suspension or as adherent cultures, and from single mouse brains. The method first describes the generation of cell mass in both suspension and adherent settings, the lysis step, followed by the successive stages of tubulin purification by polymerization-depolymerization cycles. Our method yields tubulin that can be used in experiments addressing the impact of the tubulin code on the intrinsic properties of microtubules and microtubule interactions with associated proteins.

This protocol describes tubulin purification from small/medium-scale sources such as cultured cells or single mouse brains, using polymerization and depolymerization cycles. The purified tubulin is enriched in specific isotypes or has specific posttranslational modifications and can be used in in vitro reconstitution assays to study microtubule dynamics and interactions.

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They ...

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

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

Summary

Explore More Videos

Chapters in this video

A Method for Measuring RNA N⁶-methyladenosine Modifications in Cells and Tissues

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

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

Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

Ganglioside Extraction, Purification and Profiling