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 lacking arginine and lysine. The medium must be supplemented with 10% dialyzed fetal bovine serum (FBS), 100 units/mL penicillin/streptomycin, and alanine-glutamine. Make two types of media by adding either conventional lysine and arginine (&#39;Light&#39; Medium) or lysine-8 (13C6;15N2) and arginine-10 (13C6;15N4) (&#39;Heavy&#39; Medium), respectively.</li>
		<li>Culture batches of cells in Light Medium (i.e., unlabeled) and Heavy Medium (i.e., labeled, SILAC) for at least six doublings before expansion and treatment to make sure that all proteins in the Heavy Medium culture are labeled with heavy stable isotope containing amino acids.</li>
		<li>Treat the cells for 8 h with 10 &#181;M of the proteasome inhibitor bortezomib or an equivalent volume of DMSO as a mock treatment. Wash the cells with PBS, dissociate them using 1% trypsin/EDTA, and pellet the cells.</li>
		<li>Lyse the cell pellet from one 150 cm2 culture plate per condition tested in 2 mL of ice-cold 50 mM Tris-HCl (pH = 8.2) with 0.5% sodium deoxycholate (DOC). Boil the lysate at 95 &#176;C for 5 min and sonicate for 10 min (setting &#34;H&#34; for the sonicator listed in the Table of Materials) at 4 &#176;C. We do not recommend the use of deubiquitinase inhibitors such as N-ethylmaleimide (NEM), because this may introduce unwanted protein modifications that can complicate peptide identification.</li>
	</ol>
	</li>
	<li>In vivo mouse brain tissue
	<ol>
		<li>When using in vivo tissue, lyse the tissue in an ice-cold buffer containing 100 mM Tris-HCl (pH = 8.5), 12 mM sodium DOC, and 12 mM sodium N-lauroylsarcosinate17. Sonicate the lysate for 10 min (setting &#34;H&#34; for the sonicator listed in the Table of Materials) at 4 &#176;C and boil the lysate for 5 min at 95 &#176;C.</li>
	</ol>
	</li>
	<li>Quantitate the total protein amount using a colorimetric absorbance BCA protein assay kit. The total amount of protein should be at least several milligrams for a successful diGly peptide immunoprecipitation (IP). For SILAC experiments mix the light and heavy labeled proteins in a 1:1 ratio based on the total protein amount.</li>
	<li>Reduce all proteins using 5 mM 1,4-dithiothreitol for 30 min at 50 &#176;C and subsequently alkylate them with 10 mM iodoacetamide for 15 min in the dark. Perform protein digestion with Lys-C (1:200 enzyme-to-substrate ratio) for 4 h followed by overnight digestion with trypsin (1:50 enzyme-to-substrate ratio) at 30 &#176;C or at room temperature (RT).</li>
	<li>Add trifluoroacetic acid (TFA) to the digested sample to a final concentration of 0.5% and centrifuge the sample at 10,000 x g for 10 min in order to precipitate and remove all detergent. Collect the supernatant containing the peptides for subsequent fractionation.</li>
</ol>
2. Offline peptide fractionation
<ol>
	<li>Use high pH reverse-phase (RP) C18 chromatography with polymeric stationary phase material (300 &#197;, 50 &#181;M; see Table of Materials) loaded into an empty column cartridge to fractionate the tryptic peptides. The stationary phase bed size must be adjusted to the amount of protein digest to be fractionated. Prepare an empty 6 mL column cartridge (see Table of Materials) filled with 0.5 g of stationary phase material for ~10 mg of protein digest. The protein digest to stationary phase ratio should be approximately 1:50 (w/w).</li>
	<li>Load the peptides onto the prepared column and wash the column with approximately 10 column volumes of 0.1% TFA, followed by approximately 10 column volumes of H2O.</li>
	<li>Elute the peptides into three fractions with 10 column volumes of 10 mM ammonium formate solution (pH = 10) with 7%, 13.5%, and 50% acetonitrile (AcN), respectively. Lyophilize all fractions to completeness.</li>
	<li>Use ubiquitin remnant motif (K-&#949;-GG) antibodies conjugated to protein A agarose beads for the immunoenrichment of diGly peptides. Because the exact amount of antibody per batch of beads is proprietary information and not disclosed by the manufacturer, it is recommended to use the same definition for a batch of beads as the manufacturer does in order to avoid confusion. Wash one batch of these beads 2x with PBS and split the bead slurry into six equal fractions. See Figure 1 for a detailed experimental scheme.</li>
	<li>Dissolve the three peptide fractions collected in step 2.3 in 1.4 mL of a buffer composed of 50 mM MOPS, 10 mM sodium phosphate, and 50 mM NaCl (pH = 7.2), and spin down the debris.</li>
	<li>Add the supernatants of the fractions to the diGly antibody beads and incubate for 2 h at 4 &#176;C on a rotator unit. Spin down the beads and transfer the supernatant to a fresh batch of antibody beads and incubate again for 2 h at 4 &#176;C.</li>
	<li>Store the supernatants for subsequent global proteome (GP) analysis.</li>
	<li>Transfer the beads from every fraction into a 200 &#181;L pipette tip equipped with a GF/F filter plug to retain the beads. Put the pipette tip with the beads into a 1.5 mL microcentrifuge tube equipped with a centrifuge tip adapter. Wash the beads 3x with 200 &#181;L of ice-cold IAP buffer and subsequently 3x with 200 &#181;L of ice-cold milliQ H2O. Spin down the column at 200 x g for 2 min before every wash step, but be careful not to let the column run dry. Elute the peptides using 2 cycles of 50 &#181;L of 0.15% TFA.</li>
	<li>Desalt the peptides using a C18 stage tip (essentially a 200 &#181;L pipette tip with two C18 disks) and dry them to completeness using vacuum centrifugation.</li>
</ol>
3. Nanoflow LC-MS/MS
<ol>
	<li>Perform LC-MS/MS experiments on a sensitive mass spectrometer coupled to a nanoflow LC system.</li>
	<li>Use an in-house packed 50 cm reversed-phase column with a 75 &#181;m inner diameter packed with CSH130 resin (3.5 &#181;m, 130 &#197;) and elute the peptides with a gradient of 2&#8722;28% (AcN, 0.1% FA) over 120 min at 300 nL/min. Alternatively, use commercially available LC columns with similar properties. Keep the column at 50 &#176;C using a column oven, for example (see Table of Materials).</li>
	<li>Perform the mass spectrometry analysis.
	<ol>
		<li>The mass spectrometer must be operated in data-dependent acquisition (DDA) mode. MS1 mass spectra should be collected at high resolution (e.g., 120,000), with an automated gain control (AGC) target setting of 4E5 and a maximum injection time of 50 ms in case of an Orbitrap mass spectrometer.</li>
		<li>Perform the mass spectrometry analysis in &#34;Highest Intensity First&#34; mode first. This way, the most intense ion is selected first for fragmentation, then the second highest, and so forth, using the top speed method with a total cycle time of 3 s. Subsequently, perform a second round of DDA MS analysis in &#34;Lowest Intensity First&#34; mode, so that the least intense ion is selected first, then the second lowest, and so forth. This strategy ensures optimal detection of very low abundancy peptides.</li>
		<li>Filter the precursor ions according to their charge states (2-7 charges) and monoisotopic peak assignment. Exclude previously interrogated precursors dynamically for 60 s. Isolate peptide precursors with a quadrupole mass filter set to a width of 1.6 Th.</li>
		<li>Collect MS2 spectra in the ion trap at an AGC of 7E3 with a maximum injection time of 50 ms and HCD collision energy of 30%.</li>
	</ol>
	</li>
</ol>
4. Data analysis
<ol>
	<li>Analyze the mass spectrometry raw files using an appropriate search engine such as the freely available MaxQuant software suite based on the Andromeda search engine18,19. In MaxQuant, select the default settings with a few adaptations that are indicated below. Set the enzyme specificity to trypsin, with the maximum number of missed cleavages raised to three. Set lysine with a diGly remnant (+114.04 Da), oxidation of methionine and N-terminal acetylation as variable modifications, and set carbamidomethylation of cysteine as a fixed modification.</li>
	<li>Perform database searches against a FASTA file containing protein sequences downloaded from, for example, the Uniprot repository (<a href="https://www.uniprot.org/downloads" target="_blank">https://www.uniprot.org/downloads</a>) combined with a decoy and a standard common contaminant database that is automatically provided by MaxQuant. Set the false discovery rate (FDR) to 1% and set the minimum score for modified (diGly) peptides to 40 (default value). Exclude peptides identified with a C-terminal diGly modified lysine residue from further analysis.</li>
	<li>For the quantitative analysis of SILAC experiment files, set the multiplicity to &#34;2&#34; and repeat step 4.2.</li>
	<li>Perform all downstream analyses (e.g., statistics, gene ontology analyses) with the Perseus module20 of the MaxQuant software suite.</li>
</ol>

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The authors declare no conflict of interest.

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><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 &amp;mu;m, 130 &amp;Aring;</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 (&lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;6&lt;/sub&gt;;&lt;sup&gt;15&lt;/sup&gt;N&lt;sub&gt;2&lt;/sub&gt;), Arginine-10 (&lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;6&lt;/sub&gt;;&lt;sup&gt;15&lt;/sup&gt;N&lt;sub&gt;4&lt;/sub&gt;)</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 &amp;Aring;, 50 &amp;micro;m) polymeric reversed phase particles</td><td>Agilent Technologies</td><td>PL1412-2K01</td><td></td></tr><tr><td>PTMScan Ubiquitin Remnant Motif (K-&amp;epsilon;-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...

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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 ...

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Research

JoVE Journal

Biochemistry

9.4K Views.  Erasmus University Medical Center. De post translationele modificatie van eiwitten door het kleine eiwit ubiquitine is betrokken bij vele gebeurtenissen in de cel. Deze studie presenteert een originele toevoeging aan de gereedschapskist voor eiwit alomtegenwoordigheid analyse. We gebruiken verrijkingstechnieken en massaspectrometrie om de diepe ubiquitinome te ontdekken.We hebben verschillende verbeteringen aangebracht in de analyse van diGly peptiden die afkomstig zijn van alomtegenwoordige eiwitten. Deze omvatten ruwe...

Video: Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Cancer Research

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

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

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

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

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

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

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

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

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

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate protein. The ubiquitination process occurs intracellularly in eukaryotes and regulates almost all basic cellular biological processes. Purification of ubiquitinated proteins aids the investigation of the role of ubiquitination in controlling the function of substrate proteins. Here, a step-by-step procedure to purify ubiquitinated proteins in mammalian cells is described with the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified under stringent nondenaturing and denaturing conditions. Total cellular Flag-tagged p53 protein was purified with anti-Flag antibody-conjugated agarose under nondenaturing conditions. Alternatively, total cellular His-tagged ubiquitinated protein was purified using nickel-charged resin under denaturing conditions. Ubiquitinated p53 proteins in the eluates were successfully detected with specific antibodies. Using this procedure, the ubiquitinated forms of a given protein can be efficiently purified from mammalian cells, facilitating studies on the roles of ubiquitination in regulating protein function.

The protocol describes a step-by-step method to purify ubiquitinated proteins from mammalian cells using the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified from cells under stringent nondenaturing and denaturing conditions.

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

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

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

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

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

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Immunoaffinity Based Extraction of Ubiquitinylated Peptides: A Technique to Selectively Extract Ubiquitin Tagged Peptides from Purified Peptide Fractions

Source: Bezstarosti, K. et al. <a href="https://www.jove.com/v/59079/detection-protein-ubiquitination-sites-peptide-enrichment-mass">Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry. J. Vis. Exp.</a> (2020)
This video describes a method to extract and purify ubiquitinylated peptides containing remnant di-glycine peptides from a complex peptide mixture. The presented method may help in identifying original ubiquitination sites in the protein.

Source: Bezstarosti, K. et al. Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry. J. Vis. Exp. (2020)
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Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

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