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.
<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...

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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

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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; ...

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 ...

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 ...

Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases

Ubiquitin is a small 8.6 kDa protein that is a core component of the ubiquitin-proteasome system. Consequently, it can bind to a diverse array of proteins with high specificity but low affinity. Through phage display, ubiquitin variants (UbVs) can be engineered such that they exhibit improved affinity over wildtype ubiquitin and maintain binding specificity to target proteins. Phage display utilizes a phagemid library, whereby the pIII coat protein of a filamentous M13 bacteriophage (chosen because it is displayed externally on the phage surface) is fused with UbVs. Specific residues of human wildtype ubiquitin are soft and randomized (i.e., there is a bias towards to native wildtype sequence) to generate UbVs so that deleterious changes in protein conformation are avoided while introducing the diversity necessary for promoting novel interactions with the target protein. During the phage display process, these UbVs are expressed and displayed on phage coat proteins and panned against a protein of interest. UbVs that exhibit favorable binding interactions with the target protein are retained, whereas poor binders are washed away and removed from the library pool. The retained UbVs, which are attached to the phage particle containing the UbV's corresponding phagemid, are eluted, amplified, and concentrated so that they can be panned against the same target protein in another round of phage display. Typically, up to five rounds of phage display are performed, during which a strong selection pressure is imposed against UbVs that bind weakly and/or promiscuously so that those with higher affinities are concentrated and enriched. Ultimately, UbVs that demonstrate higher specificity and/or affinity for the target protein than their wildtype counterparts are isolated and can be characterized through further experiments.

Ubiquitination is a critical protein post-translational modification, dysregulation of which has been implicated in numerous human diseases. This protocol details how phage display can be utilized to isolate novel ubiquitin variants that can bind and modulate the activity of E3 ligases that control the specificity, efficiency, and patterns of ubiquitination.

Ubiquitin is a small 8.6 kDa protein that is a core component of the ubiquitin-proteasome system. Consequently, it can bind to a diverse array of proteins ...

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.