The authors would like to thank C&#233;line Jeanty for her assistance in creation of cellular pellets with treatment of MG-132 as described in the representative results and Elise Mommaerts for her provision of E. coli pellets used in the protocol.

1. Preparation of a heavy peptide standard
<ol>
	<li>Depending on the supplier and quality of the heavy peptides purchased, the heavy peptides will need to be diluted. This protocol used the peptide sequences reported in Table 1, with the C&#8211;terminal amino acid modified to Lysine (13C615N2-lysine) or Arginine (13C615N4-arginine).
	<ol>
		<li>Mix the heavy peptides, diluting the mix with 50% acetonitrile (ACN) to create a peptide mix with each peptide at 10 &#181;M.</li>
		<li>Create aliquots of the peptide mix and store at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Sample preparation
<ol>
	<li>Sample lysis 
	NOTE: The stability of ubiquitin chains must be considered during sample preparation. Keep biological samples and buffers chilled at 4 &#176;C and add samples to ubiquitin stabilization buffer as rapidly as possible.
	<ol>
		<li>Prepare a solution of 1 M NH4HCO3 (ammonium bicarbonate) in water and adjust the pH to 8 using 1 M NaOH.</li>
		<li>Prepare a solution of 200 mM N-ethylmaleimide (NEM) in water.</li>
		<li>Prepare ubiquitin stabilization buffer using 8 M urea in 50 mM NH4HCO3/10 mM NEM. Ubiquitin stabilization buffer must be prepared freshly each time. 
		NOTE: A 5 mL solution of 8 M urea will require considerably less than 5 mL of water given the expansion of water when in a saturated solution with urea. 
		CAUTION: Do not prepare the buffer above 50 &#176;C due to the tendency of urea to form polyurea at high temperatures.</li>
		<li>Resuspend the biological sample to be investigated in ubiquitin stabilization buffer aiming for 0.5 &#181;g/&#181;L of protein and lyse the cells with an appropriate method for the sample. A sonication method consisting of three 10 s pulses with an SFX 150 Branson sonifier set at 70% intensity with 10 s breaks on ice between each pulse, was used for the cell lines in this protocol.</li>
		<li>Centrifuge sample at 18,000 x g for 10 min at 4 &#176;C in a tabletop centrifuge.</li>
		<li>Transfer supernatant to a fresh low protein binding microcentrifuge tube. Samples can be stored at -20 &#176;C at this point if needed.</li>
	</ol>
	</li>
	<li>Preparation of sample and controls
	<ol>
		<li>If the samples are frozen, mix (e.g., with a thermomixer) while they are thawed to rapidly dissolve the urea. 
		CAUTION: Do not use temperatures above 50 &#176;C due to the tendency of urea to form polyurea at high temperatures.</li>
		<li>Centrifuge sample at 18,000 x g for 10 min at 4 &#176;C in a tabletop centrifuge.</li>
		<li>Transfer 20 &#181;g of sample to a fresh low-bind microcentrifuge tube and adjust the volume to 50 &#181;L with ubiquitin stabilization buffer.</li>
		<li>Create a negative control using a normalized volume of ubiquitin stabilization buffer (50 &#181;L). In this sample, no light version of each peptide should be present, allowing observation of any light contamination arising from the spike-in heavy peptides. This negative control was also used for retention time scheduling of the PRM described in section 3.1.</li>
		<li>A positive control was also created with ubiquitin stabilization buffer and the addition of commercially available chain types to the normalized volume of ubiquitin stabilization buffer (50 &#181;L). Commonly, K63, K48, and M1 are available. In the representative results 20 ng of K48, K63, and M1 were utilized.</li>
	</ol>
	</li>
	<li>Reduction, alkylation, and digestion
	<ol>
		<li>Prepare a solution of 50 mM ammonium bicarbonate (NH4HCO3) in water and adjust the sample to pH = 8 with 1 M NaOH.</li>
		<li>A culture of E. coli grown in LB broth was centrifuged 5,000 x g for 5 min to form a pellet, which was then washed 2x with PBS before resuspension in ubiquitin stabilization buffer at 5 &#956;g/&#956;L.</li>
		<li>Add 1 &#181;g of E. coli lysate to each sample and control. This protocol uses E. coli (DH10B), but any complex lysate without ubiquitin can be used. Note that ubiquitin is common to all eukaryotes. 
		NOTE: Using an E. coli lysate matrix reduces the loss of peptides due to nonspecific adhesion to plasticware. This is particularly noticeable in the negative control or samples with a limited protein content and has a strong impact on the observation of K6312.</li>
		<li>Prepare a solution of 500 mM tris(2-carboxyethyl)phosphine (TCEP) in MS grade water (DTT can be used as an alternative).</li>
		<li>Reduce the samples and controls by the addition of TCEP to a final concentration of 50 mM. Vortex briefly before incubating for 30 min at room temperature (RT). If DTT is used, the temperature has to be elevated to 50 &#176;C. 
		CAUTION: Do not use temperatures above 50 &#176;C due to the tendency of urea to form polyurea at high temperatures.</li>
		<li>Prepare a solution of 550 mM chloroacetamide (CAA) in NH4HCO3. Store in the dark.</li>
		<li>Alkylate the samples and controls by adding CAA to a final concentration of 55 mM. Vortex briefly before incubating for 20 min at RT in the dark. 
		NOTE: CAA is used instead of iodoacetamide as the reduction agent to decrease the chance of a double carbamidomethyl modification (114.0429) on a lysine residue being mistaken for a -GG modification (114.0429)13.</li>
		<li>Add endopeptidase LysC to the samples at 1:25 (w/w) ratio to the protein content. Incubate for 3 h at 37 &#176;C.</li>
		<li>Dilute the samples and controls with 200 &#181;L of 50 mM NH4HCO3 pH = 8 (1:5 v/v).</li>
		<li>Add trypsin to the samples at 1:25 w/w ratio to the protein content. Incubate for 12 h at 37 &#176;C.</li>
		<li>Add 10% formic acid (FA) solution in MS grade water at a 1:10 ratio v/v to each sample and control sample. Check that pH is &#60;3) prior to the C18 cleanup and add more formic acid if needed.</li>
		<li>To each sample and control add 0.5 &#181;L of the heavy peptide standard created in section 1.</li>
	</ol>
	</li>
	<li>Peptide cleanup
	<ol>
		<li>Several manufacturers offer C18 cleanup tips or plates. Follow the manufacturer&#39;s instructions for the product used, and dry eluted peptides in a vacuum centrifuge, ready for MS analysis.</li>
	</ol>
	</li>
</ol>
3. Analysis by LC-MS/MS
<ol>
	<li>Execute initial PRM analysis on the heavy peptide standard in an unscheduled fashion.
	<ol>
		<li>Resuspend the dried samples in 10 &#181;L of MS loading buffer 2% ACN/0.05% trifluoroacetic acid (TFA).</li>
		<li>Equip an HPLC with a reverse-phase C18 analytical column (75 &#181;m x 15 cm, 2 &#181;m 100 &#197; C18 beads) designed for nanoflow MS. Form linear gradients exchanging MS grade water supplemented with 0.1% FA with ACN containing 0.1% FA. A sacrificial trap column (75 &#181;m x 2 cm, 3 &#181;m, 100 &#197; C18 beads) to preserve the life of the analytical column was used here but is not required.</li>
		<li>Couple the output of the analytical column to a nano-ESI source on a high-resolution mass spectrometer.</li>
		<li>Utilize an appropriate targeted proteomics method for the mass spectrometer. For example, on a Q-Exactive plus a two-experiment method, cycle between a Full MS and a PRM experiment. For the Full MS experiment, a resolution of 120,000 with an AGC target of 1e6 and 240 ms as Maximum IT was used. For the PRM experiment, the same AGC target and maximum IT was used with a lower resolution of 60,000 and an isolation window of 1.2 m/z.</li>
		<li>Inject 200 ng of the negative control onto the analytical column using the HPLC autosampler. Apply a linear gradient to the sample on the analytical column increasing the ACN concentration from 2&#8211;25% over a period of ~45 min. 
		NOTE: The 25% ACN concentration is lower than usual for proteomics due to the low hydrophilic nature of typical ubiquitin topology-characteristic peptides. If other peptides are to be quantified, a higher ACN concentration may be desired to achieve good target separation.</li>
		<li>Analyze the results of the scheduling run as described in section 4.2 to create a scheduled inclusion list.</li>
	</ol>
	</li>
	<li>Analysis of the samples
	<ol>
		<li>Run the remaining samples as described for the scheduling in 3.1 apart from the use of the scheduled inclusion list created in section 4.2. 
		NOTE: A blank must be run after the positive control to ensure no carryover from the previous run is being detected in subsequent samples.</li>
	</ol>
	</li>
</ol>
4. Software analysis
<ol>
	<li>Create an inclusion list of appropriate formatting for the utilized MS. This protocol used the open source software Skyline (version 19.1.0.193) (https://skyline.ms), which offers well-documented support and help. An inclusion list can be created for several mass spectrometers using the export isolation list facility.
	<ol>
		<li>Open a new Skyline document.</li>
		<li>Select heavy isotope modifications as appropriate to heavy topology-characteristic peptides. In Settings|Peptide Settings under the Modification tab, click on the name field to see the potential modification. Selection is based on the isotopic state of the heavy peptides purchased. In this example, synthetic peptides carrying either a heavy Lysine (13C615N2-lysine) or a heavy Arginine (13C615N4-arginine) at the C-terminus were used. Appropriate labels would be: &#8220;Label:13C(6)15N(2) (C-term K)&#8221; and &#8220;Label:13C(6)15N(4) (C-term R)&#8221;.</li>
		<li>Select Settings|Transition. Under the Filter tab include Precursor Charges 2, 3, 4.</li>
		<li>Insert peptides by selecting Edit|Insert|Peptides.</li>
		<li>Fill in the relevant fields including the topology-characteristic peptide sequence and a protein name (e.g., &#34;Chain&#8211;K63&#34;).</li>
		<li>Expand each peptide now shown in the Targets panel. Delete undesired charge states. The preferred charge state and collision energy, which may differ based on mass spectrometer used, for each topology-characteristic peptide is shown in Table 1.</li>
		<li>For each topology-characteristic peptide (except for M1 where the GG is included in the sequence) right-click on the sequence and select Modify. Add a GG as a structural modification by clicking the name field and selecting &#60;Show all&#8230;&#62; before selecting &#34;GlyGly (K)&#34;.</li>
		<li>Export the isolation list File|Export|Isolation List, select the instrument type, and set the method type to Standard. Selecting OK will open a prompt to save a *.csv file that can be used to create a PRM method.</li>
	</ol>
	</li>
	<li>Create a scheduled inclusion list based on a heavy peptide scheduling run explained here using the open source Skyline software.
	<ol>
		<li>Create a target list as described in section 4.1.</li>
		<li>Import the scheduling run in 3.1 by selecting File|Import|Results.</li>
		<li>Review the identifications. The heavy signal for each peptide is observable by clicking on the mass for each heavy peptide entry. Correct recognition of the peak is often determined automatically, but the software may require manual curation by selecting the peak using click and drag under the x-axis.</li>
		<li>Retention times selected for the heavy variants are also applied to the light versions, thus creating a schedule for the PRM. The scheduling window can be modified under Settings|Peptide Settings using the Prediction tab, by changing the Time Window.</li>
		<li>A scheduled inclusion list can now be exported using File|Export|Isolation list and selecting the appropriate instrument type and setting method type to Scheduled.</li>
	</ol>
	</li>
	<li>Analyze the data.
	<ol>
		<li>Import the samples in the same fashion as for the scheduling run analysis in section 4.2.</li>
		<li>After the complete import of all samples, curation of transitions is recommended. This is the process by which transitions with interference or poor signal-to-noise ratios are removed. An example of a chromatogram before and after curation is shown in Figure 4.</li>
		<li>Data can now be exported either by right clicking on relevant graphs and selecting Copy data or by selecting File|Export|Results.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Khoury, G. A., Baliban, R. C., Floudas, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome-wide+post-translational+modification+statistics:+frequency+analysis+and+curation+of+the+swiss-prot+database.">Proteome-wide post-translational modification statistics: frequency analysis and curation of the swiss-prot database.</a> Scientific Reports. 1, 90 (2011).</li><li>Goldstein, G., 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=Isolation+of+a+polypeptide+that+has+lymphocyte-differentiating+properties+and+is+probably+represented+universally+in+living+cells.">Isolation of a polypeptide that has lymphocyte-differentiating properties and is probably represented universally in living cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 72 (1), 11-15 (1975).</li><li>Glickman, M. H., Ciechanover, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin-proteasome+proteolytic+pathway:+Destruction+for+the+sake+of+construction.">The ubiquitin-proteasome proteolytic pathway: Destruction for the sake of construction.</a> Physiol. Rev. 82, 373-428 (2002).</li><li>Pickart, C. M., Eddins, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin:+structures,+functions,+mechanisms.">Ubiquitin: structures, functions, mechanisms.</a> Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 1695 (1-3), 55-72 (2004).</li><li>Akutsu, M., Dikic, I., Bremm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin+chain+diversity+at+a+glance.">Ubiquitin chain diversity at a glance.</a> Journal of Cell Science. 129 (5), 875-880 (2016).</li><li>Komander, D., Rape, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ubiquitin+Code.">The Ubiquitin Code.</a> Annual Review of Biochemistry. 81 (1), 203-229 (2012).</li><li>Mattern, M., Sutherland, J., Kadimisetty, K., Barrio, R., Rodriguez, 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=Using+Ubiquitin+Binders+to+Decipher+the+Ubiquitin+Code.">Using Ubiquitin Binders to Decipher the Ubiquitin Code.</a> Trends in Biochemical Sciences. 44 (7), 599-615 (2019).</li><li>Newton, K., 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=Ubiquitin+chain+editing+revealed+by+polyubiquitin+linkage-specific+antibodies.">Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies.</a> Cell. 134 (4), 668-678 (2008).</li><li>Spence, 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=Cell+cycle-regulated+modification+of+the+ribosome+by+a+variant+multiubiquitin+chain.">Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain.</a> Cell. 102 (1), 67-76 (2000).</li><li>Borr&#224;s, E., Sabid&#243;, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+targeted+proteomics?+A+concise+revision+of+targeted+acquisition+and+targeted+data+analysis+in+mass+spectrometry.">What is targeted proteomics? A concise revision of targeted acquisition and targeted data analysis in mass spectrometry.</a> PROTEOMICS. 17 (17-18), (2017).</li><li>Lesur, A., Domon, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+high-resolution+accurate+mass+spectrometry+application+to+targeted+proteomics.">Advances in high-resolution accurate mass spectrometry application to targeted proteomics.</a> PROTEOMICS. 15 (5-6), 880-890 (2015).</li><li>Longworth, J., Dittmar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Ubiquitin+Chain+Topology+by+Targeted+Mass+Spectrometry.">Assessment of Ubiquitin Chain Topology by Targeted Mass Spectrometry.</a> Methods in Molecular Biology. 1977, 25-34 (2019).</li><li>Nielsen, M. L., 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=Iodoacetamide-induced+artifact+mimics+ubiquitination+in+mass+spectrometry.">Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry.</a> Nature Methods. 5 (6), 459-460 (2008).</li><li>Tawa, N. E., Odessey, R., Goldberg, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+the+proteasome+reduce+the+accelerated+proteolysis+in+atrophying+rat+skeletal+muscles.">Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying rat skeletal muscles.</a> The Journal of Clinical Investigation. 100 (1), 197-203 (1997).</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><li>MacLean, B., 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=Effect+of+Collision+Energy+Optimization+on+the+Measurement+of+Peptides+by+Selected+Reaction+Monitoring+(SRM)+Mass+Spectrometry.">Effect of Collision Energy Optimization on the Measurement of Peptides by Selected Reaction Monitoring (SRM) Mass Spectrometry.</a> Analytical Chemistry. 82 (24), 10116-10124 (2010).</li></ol>

The authors declare no competing interests.

Analysis of the ubiquitin state within a proteome is of increasing importance to a wide variety of biological questions. The description of the ubiquitination state of a sample must focus not only on the profile of proteins being ubiquitinated but also on the topology of such ubiquitination. The assessment of this topology by targeted MS, as described here, has a role in a wide range of biological investigations.
It should be understood that the protocol outlined here provides a global topolog...

The close regulation of protein function and stability is of paramount importance, as they are major drivers of phenotypic control of biology. The function of a protein is constructed from two components: its intrinsic polypeptide sequence and any posttranslational modifications (PTMs). Various chemical PTMs have been identified including glycosylation, phosphorylation, acetylation, and methylation1. In 1975, Goldstein et al.2 identified a small protein and named it ubiquitin due to its ubiquitous nature. Ubiquitin was found to be important in protein degradation3. However, since then it has been established that the function of ubiquitin as a signaling molecule extends far beyond the regulation of protein stability. Ubiquitin signaling is involved in a wide range of other biological functions, such as protein stabilization, autophagy, cell cycle control, and protein trafficking4.
While other PTMs are generally binary (i.e., the protein is modified or left unmodified) for a given site, ubiquitin can modify a protein both as a monomer or as a polymeric chain, with the attached ubiquitin itself being ubiquitinated. Further, this polyubiquitination chain can develop in several topologies with the ubiquitination of the previous ubiquitin attaching to one of eight linkage sites5,6. Ubiquitin is transferred by a multistep enzymatic process (Figure 1) where the C-terminus of ubiquitin is linked to one of its seven lysine residues (K06, K11, K27, K29, K33, K48, or K63) or the N-terminal of the previous ubiquitin (referred to as the M1 or linear ubiquitination)5,6. This chain topology is key to the fate of the protein under modification. For example, the modification with a K48 or K11 linked chain leads to the degradation of the modified protein at the proteasome, while a linear chain is necessary for the activation of NF-kB signaling. Thus, the relative distribution of these chain topologies is relevant to a wide variety of biological questions.
The use of mass spectrometry (MS) is of particular benefit to ubiquitin chain topology analysis as it is not reliant on antibody-based or affinity-based interactions7,8, many of which have limited specificity and do not differentiate between the various chain types. A different detection possibility is using genetically modified ubiquitin mutants. Here, a specific lysine is exchanged for arginine, which cannot support the modification by ubiquitin. The lack of ubiquitin chain formation on the substrate protein is then interpreted as evidence for a specific topology9.
MS-based identification of ubiquitinated proteins is based on the fact that the C-terminus of ubiquitin contains an arginine residue in position 74 that is recognized by trypsin during the proteolytic preparation of the samples for MS analysis, separating the C-terminal double glycine. This GlyGly (-GG) remains attached to the &#949;-amino group of the lysine residue of the substrate protein. For ubiquitin topology analysis, the modification occurs on one of the seven lysine residues of ubiquitin. This creates a set of seven key peptides that carry a lysine residue that is modified by GG, specific for each of the topologies (Figure 2). For example, with K06 topology, the lysine at position 6 on the amino acid sequence will be protected from tryptic digestion with a -GG modification of 114 Da added to this lysine.
Identification of specific predetermined peptides by MS is referred to as targeted proteomics, or more specifically, targeted peptide acquisition10. Two methods have been developed depending on the performance of the mass spectrometer being used. These are selected reaction monitoring (SRM), also called multiple reaction monitoring (MRM), and parallel reaction monitoring (PRM). SRM involves the selection of transitions consisting of the precursor m/z and the product ion m/z. Conversely, PRM requires only the precursor m/z. Post selection, a full survey scan of the product ions is performed. This has the advantage that no selection of appropriate product ions for quantitation on the specific mass spectrometer is necessary prior to the measurement11. Both SRM and PRM can, depending on the instrument, be scheduled. Scheduling is the practice of assigning a time window during which a particular ion will be included for analysis, as peptides are eluting at defined retention times from the chromatographic system. Reducing the number of ions being interrogated at any given time increases the frequency of interrogation of those ions scheduled at that time, thus improving data accuracy.
In general, the application of targeted proteomics for ubiquitin topology is the same as any other targeted proteomics experiment. However, two key differences are important: First, the stability of ubiquitin chains must be considered. There are multiple potent deubiquitinating enzymes (DUBs) that rapidly degrade chains upon cell lysis. The ubiquitin-specific proteases fall into two categories, the thioesterases and metalloproteases. Most of the ubiquitin-hydrolases are thioesterases and carry a cysteine residue in their active center. By alkylating this cysteine residue, they can be inactivated. As such, the use of ubiquitin stabilization buffers containing alkylating agents, like N-ethylmaleimide (NEM), and highly denaturing chemicals, and keeping samples chilled is vital to a successful analysis. Second, unlike other targeted experiments, the peptide choice is fixed. In a typical targeted experiment, a proteotypic peptide can be selected for good chromatographic and ionization performance. For some topology-characteristic peptides such as K48 these properties are good, while for others, they are less desirable. For example, K33 chromatography in a typical reverse-phase setup is poor due to the formation of a stretched elution profile, and the poor ionization properties of the K27 peptide reduce its visibility by MS12.
In this protocol, we describe how to perform ubiquitin topology assessment of a biological sample by PRM. Example data for the procedure are presented using a perturbation of the proteasome using MG-132 inhibitor treatment in several different cell types.

<table><tbody><tr><td>Acetonitrile (ACN)</td><td>Merck</td><td>100029</td><td></td></tr><tr><td>Ammonium bicarbonate (ABC)</td><td>Fluka</td><td>9830</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>Microfuge 16</td><td></td></tr><tr><td>Chloroacetamide (CAA)</td><td>Sigma</td><td>22790</td><td></td></tr><tr><td>Eppendorf LoBind</td><td>Eppendorf</td><td>22431081</td><td></td></tr><tr><td>Formic acid (FA)</td><td>Thermo Fisher Scientific</td><td>85178</td><td></td></tr><tr><td>Heavy Peptides</td><td>JPT Peptide Technologies</td><td></td><td></td></tr><tr><td>HPLC</td><td>Dionex</td><td>Ulitimate 3000</td><td></td></tr><tr><td>LC Column</td><td>Thermo Fisher Scientific</td><td>160321</td><td></td></tr><tr><td>Lys C</td><td>Wako</td><td>125-05061</td><td></td></tr><tr><td>Mass Spectrometer</td><td>Thermo Fisher Scientific</td><td>Q-Exactive Plus</td><td></td></tr><tr><td>N-ethylmaleimide (NEM)</td><td>ACROS Organics</td><td>156100050</td><td></td></tr><tr><td>Positive Control Chain K48</td><td>Boston Biochem</td><td>UC-240</td><td></td></tr><tr><td>Positive Control Chain K63</td><td>Boston Biochem</td><td>UC-340-100</td><td></td></tr><tr><td>Positive Control Chain M1</td><td>Boston Biochem</td><td>UC-710B-025</td><td></td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>Sigma</td><td>S5881</td><td></td></tr><tr><td>Sonifier</td><td>Branson sonifier</td><td>SFX 150</td><td></td></tr><tr><td>Thermomixer</td><td>Eppendorf</td><td>Thermomixer Comfort</td><td></td></tr><tr><td>Trifluoroacetic acid (TFA)</td><td>Sigam</td><td>T6508</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine (TCEP)</td><td>Thermo Fisher Scientific</td><td>77720</td><td></td></tr><tr><td>Trypsin</td><td>Promega</td><td>V1511A</td><td></td></tr><tr><td>Urea</td><td>Sigma</td><td>51456</td><td></td></tr><tr><td>Waters &amp;mu;Elution C&lt;sub&gt;18&lt;/sub&gt; plates</td><td>Waters</td><td>186002318</td><td></td></tr></tbody></table>

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.

To demonstrate the use of a ubiquitin chain analysis by PRM, three cell lines were selected: a mouse melanoma cell line B16, and the two common human cell lines A549 (adenocarcinomic alveolar basal epithelial cells) and HeLa (cervical cancer cells). These cultures grew to midexponential phase in appropriate media before being treated with 0, 10, or 100 mM MG-132 for 4 h prior to harvest. MG-132 is a proteasome inhibitor preventing the degradation of ubiquitin-conjugated proteins by the proteasome14</sup...

Watch this Scientific Journal Video about Ubiquitin Chain Analysis by Parallel Reaction Monitoring at JoVE.com

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

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 ...

ubiquitin-chain-analysis-by-parallel-reaction-monitoring

Research

JoVE Journal

Biochemistry

3.5K Views.  Luxembourg Institute of Health. 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.

Video: Ubiquitin Chain Analysis by Parallel Reaction Monitoring

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 ...

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

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

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

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

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

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, ...

Assessing DUB Variants in Neurodevelopmental Disorders Using a Cleavage Assay

Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay

Neurodevelopmental disorders (NDDs) are associated with impairments in nervous system function but often remain poorly understood at the molecular level. Discrete disorders caused by single genes provide models to investigate mechanisms driving atypical neurodevelopment. Variants of genes encoding deubiquitylating enzyme (DUB) family proteins are associated with several NDDs, but there is a need to determine the pathogenic mechanisms of disorders driven by these gene variants. The impact of gene variants on DUB activity can be experimentally determined using a substrate-independent in vitro ubiquitin cleavage assay. This assay does not require knowledge of downstream substrates to directly measure catalytic activity. Here, the protocol for determining the impact of gene variants on enzymatic activity is modeled using the DUB Ubiquitin Specific Protease 27, X-linked (USP27X), which is mutated in X-linked intellectual disability 105 (XLID105). This experimental pipeline can be used to clarify the mechanisms underlying neurodevelopmental disorders driven by variants in DUB genes.

This protocol explains how to measure the effect of genetic variation associated with neurodevelopmental disorders on deubiquitylating enzyme activity by combining recombinant protein purification with ubiquitin chain cleavage assays.

Neurodevelopmental disorders (NDDs) are associated with impairments in nervous system function but often remain poorly understood at the molecular level. ...

Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain

The ubiquitin-proteasome system is a key regulator of protein degradation and a variety of other cellular processes in eukaryotes. In the brain, increases in ubiquitin-proteasome activity are critical for synaptic plasticity and memory formation and aberrant changes in this system are associated with a variety of neurological, neurodegenerative and psychiatric disorders. One of the issues in studying ubiquitin-proteasome functioning in the brain is that it is present in all cellular compartments, in which the protein targets, functional role and mechanisms of regulation can vary widely. As a result, the ability to directly compare brain ubiquitin protein targeting and proteasome catalytic activity in different subcellular compartments within the same animal is critical for fully understanding how the UPS contributes to synaptic plasticity, memory and disease. The method described here allows collection of nuclear, cytoplasmic and crude synaptic fractions from the same rodent (rat) brain, followed by simultaneous quantification of proteasome catalytic activity (indirectly, providing activity of the proteasome core only) and linkage-specific ubiquitin protein tagging. Thus, the method can be used to directly compare subcellular changes in ubiquitin-proteasome activity in different brain regions in the same animal during synaptic plasticity, memory formation and different disease states. This method can also be used to assess the subcellular distribution and function of other proteins within the same animal.

This protocol is designed to efficiently quantify ubiquitin-proteasome system (UPS) activity in different cellular compartments of the rodent brain. Users are able to examine UPS functioning in nuclear, cytoplasmic and synaptic fractions in the same animal, reducing the amount of time and number of animals needed to perform these complex analyses.

The ubiquitin-proteasome system is a key regulator of protein degradation and a variety of other cellular processes in eukaryotes. In the brain, increases ...

Neuroscience

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, ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Virtual Screening and Validation of UCHL3 Inhibitors Using Molecular Docking

Screening Traditional Chinese Medicine Compounds for Inhibiting UCHL3 Activity Based on Molecular Docking and Deubiquitinating Enzyme Probe Technology

Deubiquitinating enzymes&#160;(DUBs) play a pivotal role in modulating ubiquitination homeostasis, with UCHL3 being an archetypal cysteine DUB intricately involved in a myriad of physiological and pathological processes. Therefore, developing small molecule inhibitors targeting Ubiquitin C-Terminal Hydrolase L3 (UCHL3) is of great significance. This protocol aims to establish a process for virtual screening and in vitro validation of small molecule inhibitors of cysteine DUB represented by UCHL3. Firstly, potential inhibitors of UCHL3 are virtually screened using molecular docking technology, and the interaction between drugs and protein targets is visualized. Subsequently, the effectiveness of the screened drug, Danshensu, is verified through in vitro activity inhibition assays. Ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) and hemagglutinin-ubiquitin-vinyl sulfone (HA-Ub-VS) are used as probes for in vitro activity testing, as they can competitively bind to DUB with small molecule inhibitors to assess the activity of UCHL3. The results indicate that Danshensu has a good binding affinity with UCHL3 in molecular docking, and it can competitively inhibit the activity of UCHL3 with HA-Ub-VS. These findings provide important references for further research and development of therapeutic drugs targeting UCHL3.

The experiment used here shows a method of molecular docking combined with probe technologies to predict and validate the interaction between small molecules of traditional Chinese medicine and protein targets.

Deubiquitinating enzymes&#160;(DUBs) play a pivotal role in modulating ubiquitination homeostasis, with UCHL3 being an archetypal cysteine DUB intricately ...

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Summary

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Chapters in this video

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

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

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

Detection of Protein Ubiquitination

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

Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay

Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain

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

In Vitro Analysis of E3 Ubiquitin Ligase Function

Screening Traditional Chinese Medicine Compounds for Inhibiting UCHL3 Activity Based on Molecular Docking and Deubiquitinating Enzyme Probe Technology