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><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 &amp;micro;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 &amp;micro;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

Universidad de Cartagena UDEC

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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

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 ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

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

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

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

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Biology

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The protocol outlined here takes advantage of the di-glycine (-GG) modification left after the tryptic digestion of ubiquitin incorporated in a chain. By quantifying these topology-characteristic peptides the relative abundance of each ubiquitin chain topology can be determined. The steps required to quantify these peptides by a parallel reaction monitoring experiment are reported taking into consideration the stabilization of ubiquitin chains. Preparation of heavy controls, cell lysis, and digestion are described along with the appropriate mass spectrometer setup and data analysis workflow. An example data set with perturbations in ubiquitin topology is presented, accompanied by examples of how optimization of the protocol can affect results. By following the steps outlined, a user will be able to perform a global assessment of the ubiquitin topology landscape within their biological context.

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

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

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

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

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

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

Measuring Ubiquitylated, SUMOylated, and ADP-Ribosylated DNA-Protein Crosslinks

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

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

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

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

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

Histone Proteomic Analysis and Post-Translational Modification Characterization

Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as posttranslational modifications (PTMs). Identifying the position and nature of each PTM or pattern of PTMs in reference to external or genetic factors allows this information to be statistically correlated with biological responses such as DNA transcription, replication, or repair. In the present work, a high-throughput analytical protocol for the detection of histone PTMs from biological samples is described. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) enables the separation and PTM assignment of the most biologically relevant modifications in a single analysis. The described approach takes advantage of recent developments in dependent data acquisition (DDA) using parallel accumulation in the mobility trap, followed by sequential fragmentation and collision-induced dissociation. Histone PTMs are confidently assigned based on their retention time, mobility, and fragmentation pattern.

Author Spotlight: Enhanced Histone PTM Isomer Identification Through LC-TIMS-ToF MS/MS and PASEF

An analytical workflow based on liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) for high confidence and highly reproducible "bottom-up" analysis of histone modifications and identification based on principal parameters (retention time [RT], collision cross section [CCS], and accurate mass-to-charge [m/z] ratio).

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as ...

Chemistry

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, techniques for studying the regulatory mechanism of this system are essential to elucidating the cellular and molecular processes of the aforementioned diseases. The use of hemagglutinin derived ubiquitin probes outlined in this paper serves as a valuable tool for the study of this system. This paper details a method that enables the user to perform assays that give a direct visualization of deubiquitinating enzyme activity. Deubiquitinating enzymes control proteasomal degradation and share functional homology at their active sites, which allows the user to investigate the activity of multiple enzymes in one assay. Lysates are obtained through gentle mechanical cell disruption and incubated with active site directed probes. Functional enzymes are tagged with the probes while inactive enzymes remain unbound. By running this assay, the user obtains information on both the activity and potential expression of multiple deubiquitinating enzymes in a fast and easy manner. The current method is significantly more efficient than using individual antibodies for the predicted one hundred deubiquitinating enzymes in the human cell.

The current protocol details a method for measuring the activity of functionally homologous deubiquitinating enzymes. Specialized probes covalently modify the enzyme and allow for detection. This method holds the potential to identify new therapeutic targets.

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into protein function. Despite the importance of multi-PTM enrichment studies, few studies investigate more than one PTM at a time, due partially to the expenses, time, and large protein quantities required to perform multiple global proteomic analysis of PTMs. The &#34;one-pot&#34; affinity enrichment detailed in this protocol overcomes these barriers by permitting the simultaneous identification and quantification of peptides with lysine residues containing acetylation and succinylation PTMs with low amounts of sample input. The protocol involves preparation of protein lysate from mouse livers of SIRT5 knockout mice, performance of trypsin digestion, enrichment for PTMs, and performance of mass spectrometric analysis using a data-independent acquisition (DIA) workflow. Because this workflow allows for the enrichment of two PTMs from the same sample simultaneously, it provides a practical tool to study PTM crosstalk without requiring large amounts of samples, and it greatly reduces the time required for sample preparation, data acquisition, and analysis. The DIA component of the workflow provides comprehensive PTM-specific information. This is particularly important when studying PTM site localization, as DIA provides comprehensive sets of fragment ions that can be computationally deciphered to differentiate between different PTM localization isoforms.

This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into ...