Name: ubiquitin chain analysis by parallel reaction monitoring
Uploaded: 2020-06-17T13:00:00
Description: Watch this Scientific Journal Video about Ubiquitin Chain Analysis by Parallel Reaction Monitoring at JoVE.com

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) t...

<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>

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 &#956;Elution C18 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 ...

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