This work is part of the project &#34;Proteins at Work&#34;, a program of the Netherlands Proteomics Centre financed by The Netherlands Organization for Scientific Research (NWO) as part of the National Roadmap Large-Scale Research Facilities (project number 184.032.201).

All methods described here have been approved by the Institutional Animal Care and Use Committee (EDC) of Erasmus MC.
1. Sample preparation
<ol>
	<li>Cultured cells
	<ol>
		<li>Select a cell line of interest (e.g., HeLa or osteosarcoma [U2OS] cells) and grow the cells in Dulbecco&#39;s Minimal Eagle Medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS) and 100 units/mL penicillin/streptomycin.</li>
		<li>For quantitative proteomics experiments, culture cells in DMEM l...

<ol>
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J., Urb&#233;, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin:+Same+molecule,+different+degradation+pathways.">Ubiquitin: Same molecule, different degradation pathways.</a> Cell. 143, 682-685 (2010).</li><li>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.">The ubiquitin-proteasome proteolytic pathway.</a> Cell. 79 (1), 13-21 (1995).</li><li>Ohtake, F., Tsuchiya, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JB+special+review+-+Recent+topics+in+ubiquitin-proteasome+system+and+autophagy:+The+emerging+complexity+of+ubiquitin+architecture.">JB special review - Recent topics in ubiquitin-proteasome system and autophagy: The emerging complexity of ubiquitin architecture.</a> Journal of Biochemistry. 161 (2), 125-133 (2017).</li><li>Bergink, S., Jentsch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+ubiquitin+and+SUMO+modifications+in+DNA+repair.">Principles of ubiquitin and SUMO modifications in DNA repair.</a> Nature. 458 (7237), 461-467 (2009).</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>Michel, M. A., Swatek, K. N., Hospenthal, M. K., Komander, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin+Linkage-Specific+Affimers+Reveal+Insights+into+K6-Linked+Ubiquitin+Signaling.">Ubiquitin Linkage-Specific Affimers Reveal Insights into K6-Linked Ubiquitin Signaling.</a> Molecular Cell. 68 (1), 233-246 (2017).</li><li>Swatek, K. N., 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=Insights+into+ubiquitin+chain+architecture+using+Ub-clipping.">Insights into ubiquitin chain architecture using Ub-clipping.</a> Nature. 572, 533-537 (2019).</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>Wagner, S. A., 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=Proteomic+Analyses+Reveal+Divergent+Ubiquitylation+Site+Patterns+in+Murine+Tissues.">Proteomic Analyses Reveal Divergent Ubiquitylation Site Patterns in Murine Tissues.</a> Molecular &amp; Cellular Proteomics. 11 (12), 1578-1585 (2012).</li><li>Iesmantavicius, V., Weinert, B. T., Choudhary, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergence+of+Ubiquitylation+and+Phosphorylation+Signaling+in+Rapamycin-treated+Yeast+Cells.">Convergence of Ubiquitylation and Phosphorylation Signaling in Rapamycin-treated Yeast Cells.</a> Molecular &amp; Cellular Proteomics. 13 (8), 1979-1992 (2014).</li><li>Elia, A. E. H., 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=Quantitative+Proteomic+Atlas+of+Ubiquitination+and+Acetylation+in+the+DNA+Damage+Response.">Quantitative Proteomic Atlas of Ubiquitination and Acetylation in the DNA Damage Response.</a> Molecular Cell. 59 (5), 867-881 (2015).</li><li>Wagner, S. A., 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+Proteome-wide,+Quantitative+Survey+of+In+Vivo+Ubiquitylation+Sites+Reveals+Widespread+Regulatory+Roles.">A Proteome-wide, Quantitative Survey of In Vivo Ubiquitylation Sites Reveals Widespread Regulatory Roles.</a> Molecular &amp; Cellular Proteomics. 10 (10), M111.013284 (2011).</li><li>Udeshi, N. D., 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=Methods+for+Quantification+of+in+vivo+Changes+in+Protein+Ubiquitination+following+Proteasome+and+Deubiquitinase+Inhibition.">Methods for Quantification of in vivo Changes in Protein Ubiquitination following Proteasome and Deubiquitinase Inhibition.</a> Molecular &amp; Cellular Proteomics. 11, 148-159 (2012).</li><li>Sap, K. A., Bezstarosti, K., Dekkers, D. H., Voets, O., Demmers, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+proteomics+reveals+extensive+remodeling+of+the+ubiquitinome+after+perturbation+of+the+proteasome+by+dsRNA+mediated+subunit+knockdown.">Quantitative proteomics reveals extensive remodeling of the ubiquitinome after perturbation of the proteasome by dsRNA mediated subunit knockdown.</a> Journal of Proteome Research. 16 (8), 2848-2862 (2017).</li><li>Xu, G., Paige, J. S., Jaffrey, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+lysine+ubiquitination+by+ubiquitin+remnant+immunoaffinity+profiling.">Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling.</a> Nature Biotechnology. 28 (8), 868-873 (2010).</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>Wakabayashi, 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=Phosphoproteome+analysis+of+formalin-fixed+and+paraffin-embedded+tissue+sections+mounted+on+microscope+slides.">Phosphoproteome analysis of formalin-fixed and paraffin-embedded tissue sections mounted on microscope slides.</a> Journal of Proteome Research. 13 (2), 915-924 (2014).</li><li>Cox, J., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MaxQuant+enables+high+peptide+identification+rates,+individualized+p.p.b.-range+mass+accuracies+and+proteome-wide+protein+quantification.">MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.</a> Nature Biotechnology. 26 (12), 1367-1372 (2008).</li><li>Tyanova, S., Temu, T., Cox, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MaxQuant+computational+platform+for+mass+spectrometry-based+shotgun+proteomics.">The MaxQuant computational platform for mass spectrometry-based shotgun proteomics.</a> Nature Protocols. 11 (12), 2301-2319 (2016).</li><li>Tyanova, S., 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=The+Perseus+computational+platform+for+comprehensive+analysis+of+(prote)omics+data.">The Perseus computational platform for comprehensive analysis of (prote)omics data.</a> Nature Methods. 13 (June), 731-740 (2016).</li><li>Rose, C. 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=Highly+Multiplexed+Quantitative+Mass+Spectrometry+Analysis+of+Ubiquitylomes.">Highly Multiplexed Quantitative Mass Spectrometry Analysis of Ubiquitylomes.</a> Cell Systems. 3 (4), 395-403 (2016).</li><li>Kaiser, S. E., 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=Protein+standard+absolute+quantification+(PSAQ)+method+for+the+measurement+of+cellular+ubiquitin+pools.">Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools.</a> Nature Methods. 8 (8), 691-696 (2011).</li><li>Van Der Wal, 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=Improvement+of+ubiquitylation+site+detection+by+Orbitrap+mass+spectrometry.">Improvement of ubiquitylation site detection by Orbitrap mass spectrometry.</a> Journal of Proteomics. (July), (2017).</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></ol>

The protocol described here was applied to samples from various biological sources, such as cultured cells and in vivo tissue. In all cases we identified thousands of diGly peptides, provided that the total protein input amount was at least 1 mg. The enrichment using specific antibodies is highly efficient, given that only at most 100-150 very low abundant diGly peptides were identified from whole cell lysates if no enrichment procedures for ubiquitinated proteins or diGly peptides were applied. Obviously, sensitive mass...

The conjugation of ubiquitin to proteins marks them for degradation by the proteasome and is a crucial process in proteostasis. The C-terminal carboxyl group of ubiquitin forms an isopeptide bond with the lysine &#949;-amino group of the target protein1,2. In addition, ubiquitin can be attached to other ubiquitin modules, resulting in the formation of homogeneous (i.e., K48 or K11) or branched (i.e., heterogeneous or mixed) polyubiquitin structures1,3. The most well-known function of ubiquitin is its role in proteasomal degradation, mediated by K48-linked polyubiquitin. However, it has become clear that both mono- as well as polyubiquitination also play roles in many processes that are independent of degradation by the proteasome. For instance, K63-linked chains have nondegradative roles in intracellular trafficking, lysosomal degradation, kinase signaling, and the DNA damage response4,5. The other six linkage types are less abundant and their roles are still largely enigmatic, although first indications about their functions in the cell are emerging, largely because of the development of novel tools to enable linkage-specific detection6,7.
Mass spectrometry has become an indispensable tool for proteome analyses and nowadays thousands of different proteins from virtually any biological source can be identified in a single experiment. An additional layer of complexity is presented by posttranslational modifications (PTMs) of proteins (e.g., phosphorylation, methylation, acetylation, and ubiquitination) which can modulate protein activity. Large-scale identification of PTM-bearing proteins has also been made possible by developments in the mass spectrometry field. The relatively low stoichiometry of peptides bearing PTMs compared to their unmodified counterparts presents a technical challenge and biochemical enrichment steps are generally necessary prior to the mass spectrometry analysis. Over the past two decades, several different specific enrichment methods have been developed for the analysis of PTMs.
Because of the multifaceted roles of protein ubiquitination in the cell, there is a great demand for the development of analytical methods for the detection of ubiquitination sites on proteins8. The application of mass spectrometric methods has led to an explosion of the number of identified ubiquitination sites in fruit fly, mouse, human, and yeast proteins9,10,11,12,13,14. A major step was presented by the development of immunoprecipitation based enrichment strategies at the peptide level using antibodies directed against the K-&#949;-GG remnant motif (also referred to as &#39;diglycine&#39; or &#39;diGly&#39;). These diGly peptides are produced upon digestion of ubiquitinated proteins using trypsin as the protease15,16.
Here, we present an optimized workflow to enrich for diGly peptides using immunopurification and subsequent detection by Orbitrap mass spectrometry. Using a combination of several modifications of existing workflows, especially in the sample preparation and mass spectrometry stages, we can now routinely identify more than 23,000 diGly peptides from a single sample of HeLa cells treated with a proteasome inhibitor and ~10,000 from untreated HeLa cells. We have applied this protocol to lysates from both unlabeled and stable isotope labeling with amino acids in cell culture (SILAC) labeled HeLa cells as well as to endogenous samples such as brain tissue.
This workflow presents a valuable addition to the repertoire of tools for the analysis of ubiquitination sites in order to uncover the deep ubiquitinome. The following protocol describes all steps of the workflow in detail.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,4-Dithioerythritol</td>
			<td>Sigma-Aldrich</td>
			<td>D8255</td>
			<td></td>
		</tr>
		<tr>
			<td>3M Empore C18 Octadecyl disks</td>
			<td>Supelco</td>
			<td>66883-U</td>
			<td>product discontinued at Supelco; CDS Analytical is the new manufacturer (https://www.cdsanalytical.com/empore)</td>
		</tr>
		<tr>
			<td>Ammonium formate</td>
			<td>Sigma-Aldrich</td>
			<td>70221</td>
			<td></td>
		</tr>
		<tr>
			<td>Bortezomib</td>
			<td>UBPbio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CSH130 resin, 3.5 &#956;m, 130 &#197;</td>
			<td>Waters</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>34869</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EASY-nanoLC 1200</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GF/F filter plug</td>
			<td>Whatman</td>
			<td>1825-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma-Aldrich</td>
			<td>I6125</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysine, Arginine</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lysine-8 (13C6;15N2), Arginine-10 (13C6;15N4)</td>
			<td>Cambridge Isotope Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lysyl Endopeptidase(LysC)</td>
			<td>Wako Pure Chemicals</td>
			<td>129-02541</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoLC oven</td>
			<td>MPI design, MS Wil GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N-Lauroylsarcosine sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>L-5125</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbitrap Fusion Lumos mass spectrometer</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>ThermoFisher / Pierce</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>PLRP-S (300 &#197;, 50 &#181;m) polymeric reversed phase particles</td>
			<td>Agilent Technologies</td>
			<td>PL1412-2K01</td>
			<td></td>
		</tr>
		<tr>
			<td>PTMScan Ubiquitin Remnant Motif (K-&#949;-GG) Kit</td>
			<td>Cell Signaling Technologies</td>
			<td>5562</td>
			<td></td>
		</tr>
		<tr>
			<td>Sep-Pak tC18 6 cc Vac Cartridge</td>
			<td>Waters</td>
			<td>WAT036790</td>
			<td>Remove the tC18 material from the cartridge before filling the cartridge with PLRP-S</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>30970</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>Sigma-Aldrich</td>
			<td>T6066</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>T5941</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin, TPCK Treated</td>
			<td>ThermoFisher</td>
			<td>20233</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Ubiquitinated proteins leave a 114.04 Da diglycine remnant on the target lysine residue when the proteins are digested with trypsin. The mass difference caused by this motif was used to unambiguously recognize the site of ubiquitination in a mass spectrometry experiment. The strategy that we describe here is a state-of-the-art method for the enrichment and subsequent identification of diGly peptides by nanoflow LC-MS/MS (Figure 1A). In this s...

Watch this Scientific Journal Video about Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry at JoVE.com

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

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 ...

The post translational modification of proteins by the small protein ubiquitin is involved in many events in the cell. This study presents an original addition to the toolbox for protein ubiquitination analysis. We use enrichment techniques and mass spectrometry to uncover the deep ubiquitinome.We have made several improvements to the analysis of diGly peptides that originate from ubiquitinated proteins. These include crude peptide fractionation prior to enrichment, and the application of more advanced peptide fragmentation settings in the Orbitrap. Altogether this results in a larger coverage of the ubiquitinome.Several aspects of the protocol are difficult to describe in words. Visualization of some of the key steps is essential to be able to repeat these analyses by different operator in a different lab. The procedure will be demonstrated by Karel Bezstarosti, who is a senior technician in my lab.Begin by preparing cultured cells or mouse brain tissue for the experiment. If working with cells, lyse the cell pellet from one 150 centimeter squared culture plate in two milliliters of ice cold, 50 millimolar Tris HCL with 0.5%sodium deoxycholate. Boil the lysate at 95 degrees Celsius for five minutes.Then sonicate it at four degrees Celsius for 10 minutes. If using in vivo mouse brain tissue, lyse it in an ice cold buffer containing 100 millimolar Tris HCL, 12 millimolar sodium deoxycholate and 12 millimolar sodium N-lauroylsarcosinate. Sonicate the lysate for 10 minutes at four degrees Celsius.Then boil it for five minutes at 95 degrees Celsius. Next, quantitate the total protein amount using a calorimetric absorbance BCA protein assay kit, which should be at least several milligrams for successful diGly peptide immunoprecipitation. For SILAC experiments, mix the light and heavy labeled proteins in a one to one ratio based on the total protein amount.Reduce all proteins with five millimolar DTT for 30 minutes at 50 degrees Celsius. And subsequently alkylate them with 10 millimolar iodoacetamide for 15 minutes in the dark. Then perform protein digestion with Lys-C for four hours.And trypsin digestion overnight at 30 degrees Celsius, or at room temperature. On the next day, divide the sample into two two milliliter Eppendorf tubes and add TFA to the digested sample to a final concentration of 0.5%Centrifuge it at 10, 000 times G for 10 minutes to precipitate and remove all detergent. Collect the peptide containing supernatant for subsequent fractionation.Use high pH reverse phase C18 chromatography to fractionate the tryptic peptides. Prepare an empty six milliliter column cartridge filled with 0.5 grams of stationary phase material for about 10 milligrams of protein digest. Load the peptides onto the prepared column and wash it with approximately 10 volumes of 0.1%TFA, followed by 10 volumes of water.Elute the peptides into three fractions using 10 column volumes of 10 millimolar ammonium formate with seven, 13.5 and 50%acetonitrile, respectively. Then, litholyse all fractions. One batch of ubiquitin remnant motif antibodies conjugated to protein A agarose bead slurry is split into six equal fractions.Dissolve the three peptide fractions according to the manuscript directions and spin down the debris. Add the supernatants of the three fractions to the bead slurry while keeping the other three fractions on ice. And incubate them for two hours at four degrees Celsius on a rotator unit.Then, spin down the beads. Transfer the supernatant to a fresh batch of beads. And repeat the incubation with the remaining three bead slurry fractions.Store the supernatants for subsequent global proteome analysis. And transfer the beads to 200 microliter pipette tips equipped with a GFF filter plug. Put the tips into 1.5 milliliter tubes equipped with a centrifuge tip adaptor and wash the beads three times with 200 microliters of ice cold IAP buffer, followed by three washes with ice cold purified water.Spin down the columns at 200 times G for two minutes between washes, making sure not to let the column run dry. After the final wash, elute the peptides with two cycles at 50 microliters of 0.15%TFA. Desalt the peptides with a C18 stage tip and dry them with vacuum centrifugation.Perform LC-MS/MS experiments on a sensitive mass spectrometer coupled to a nanoflow LC system. The column is set up according to manuscript directions and kept at 50 degrees Celsius. Operate the mass spectrometer in data dependent acquisition mode.Collect the MS1 mass spectra at high resolution with an automated gain controlled target setting of 4E5 and a maximum injection time of 50 milliseconds. Perform the mass spectrometry analysis in most intense first mode, using the top speed method with a total cycle time of three seconds. Then perform a second round of DDA MS analysis in least intense first mode, which will ensure optimal detection of low abundance peptides.Filter the precursor ions according to their charge states and monoisotopic peak assignment. And exclude previously interrogated precursors dynamically for 60 seconds. Isolate peptide precursors with a quadrupole mass filter set to a width of 1.6 Thomson.Then collect MS2 spectra in the ion trap at an automated gain control of 7E3 with a maximum injection time of 50 milliseconds and HCD collision energy of 30%Analyze the mass spectrometry raw files using an appropriate search engine, such as the freely available MaxQuant software suite based on the Andromeda search engine. Open MaxQuant and select the appropriate raw data files. Set the number of processors and click on the group specific parameters tab.Select digestion, and allow for three missed cleavages. Then select modifications and add diGly to the variable modifications. Select the global parameters tab and add the correct protein sequence database.Leave the other settings as default and press start to perform the database search. For quantitative analysis of SILAC experiment files, set the multiplicity to two, and select the heavy amino acid labels. When the search is finished, import the text files into Perseus.This protocol was used to identify ubiquitination sites in proteins from cultured cells and in vivo material by detecting diGly peptides with nanoflow LC-MS/MS. Several improvements were made to the existing protocol which resulted in higher numbers of detected diGly peptides. A crude fractionation into three fractions was performed prior to immunoprecipitation.One of the fractions contains ubiquitin's own K48 modified tryptic diGly peptide, which is characterized by a broad peak in the LC chromatogram. Furthermore, the peptide fragmentation regime was adjusted to combine the LC-MS runs with the highest first and lowest first fragmentation regimes in the data analysis procedure, which produced more than 4, 000 additional unique diGly peptides. More than 23, 000 diGly peptides can be routinely identified from a single sample of HeLa cells treated with a proteasome inhibitor.Of all diGly peptides identified over three biological replicate screens, more than 9, 000 were present in all three, while more than 17, 000 were present in at least two out of three replicates. The number of peptide identifications is highly contingent on the amount of input material. An approximate number of identified diGly peptides can be expected depending on the starting material, but these numbers are only estimations and will also depend on the type of mass spectrometer used.When carrying out this protocol, prior experience with mass spectrometry based proteomics methods and handling and analyzing minute sample amounts would certainly be advantageous. The MaxQuant software could be replaced by any other database searching algorithm. Also a different type of mass spectrometer may be used.The results in terms of peptide identifications may slightly differ, but this is typical for any mass spectrometry based proteomics assay. Ubiquitin is important in progress of mediated protein degradation, but also plays a role in many other processes in the cell. Deeper knowledge of ubiquitin as a post translational modification is crucial to better understand its function.

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Research

JoVE Journal

Biochemistry

9.2K vistas.  Erasmus University Medical Center. La modificación post traslacional de proteínas por la pequeña proteína ubiquitina participa en muchos eventos en la célula. Este estudio presenta una adición original a la caja de herramientas para el análisis de la ubiquitinación de proteínas. Utilizamos técnicas de enriquecimiento y espectrometría de masas para descubrir el ubiquitinoma profundo.Hemos realizado varias mejoras en el análisis de péptidos diGly que se originan a partir de proteínas ubiquitinadas. Estos inc...