identification of the ubiquitinated proteins

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IDENTIFICATION OF THE UBIQUITINATED PROTEINS

Olesya O. Panasenko*

Department of Microbiology and Molecular Medicine, Faculty of Medicine, Institute of Genetics and Genomics of Geneva, University of Geneva, Switzerland

Correspondance: Dr Olesya O. Panasenko Dpt. Microbiologie et Médecine Moléculaire CMU, 1 rue Michel Servet, 1211 Geneva 4 Tel: 0041 22 3795516 Fax: 0041 22 3795702 [email protected]

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IDENTIFICATION OF THE UBIQUITINATED PROTEINS

Ubiquitin. Types of ubiquitination Ubiquitin is 8.5 kDa polypeptide consisting of 76 amino acids. Ubiquitin is highly conserved among eukaryotes and it can be covalently attached to target Lys either as a monomer or as a Lys-linked polymer. Conjugation of ubiquitin to intracellular proteins can alter the property of its target in a variety of ways from localization, activity, partner binding, to their selective degradation. In the yeast Saccharomyces Cerevisiae ubiquitin is coded by 4 genes UBI1-4 [1]. UBI1, UBI2 and UBI3 encode hybrid proteins in which ubiquitin is fused to the large ribosomal proteins Rpl40A and Rpl40B or to the small ribosomal protein Rps31, respectively. The fourth gene, UBI4, encodes a polyubiquitin precursor comprised of 5 head-to-tail ubiquitin repeats. This gene is strongly inducible by different stresses as high temperatures, starvation, and others. In humans ubiquitin is also encoded by 4 different genes. UBA52 and RPS27A genes code for a single copy of ubiquitin fused to the ribosomal proteins L40 and S27A, respectively. UBB and UBC genes code for a polyubiquitin precursor with head-to-tail repeats. The number of repeats differs between species. At the protein level, it is not possible to determine from which of the 4 genes ubiquitin chain was derived. Ubiquitination is a highly regulated process that involves the consecutive actions of E1, E2 and E3 enzymes (Fig. 1). E1, ubiquitin-activating enzyme, activates ubiquitin by forming a thiol ester link between the carboxy terminus Gly-76 of ubiquitin and the Cys of E1 in an ATP-dependent manner. The activated ubiquitin is then transferred to an E2 ubiquitinconjugating enzyme, also through a thiol ester bond. E2 interacts with E3 ubiquitin ligase, that recognizes the substrate. Finally, ubiquitin can be transferred to its target, forming a covalent isopeptide bond between the carboxyl terminus Gly76 of ubiquitin and a primary amine (usually the ε-amino group of Lys) of the target protein. In rare cases ubiquitin may be conjugated to Cys, Ser or Thr residues of target proteins [2]. Human genome encodes 2 E1s, about 50 E2s and more that 600 E3s [3], while yeast has 1 E1s, 13 E2s and about 100 E3s [4]. Ubiquitination may occur once resulting in monoubiquitination; several times but on different Lys of the substrate, resulting in multi-monoubiquitination; and finally, several times but on the same Lys of the substrate, resulting in polyubiquitination (Fig. 2). Monoubiquitination and multi-monoubiquitination are involved in modulation of protein activity, localization, and interactions. There are 7 lysines in ubiquitin molecule (Lys6, 11, 27, 29, 33, 48, 63) and N2

terminal methione (Met1) that can be ubiquitinated. Polyubiquitin chain can be linked via any one of the 7 Lys or the N-terminal Met. Thereby, at least 8 types of ubiquitin chains may be formed, which are molecularly identical but structurally very distinct and lead to different outcomes through interaction with distinct Ub-receptors [5-7]. Thus, Lys6-linked chains may be involved in DNA repair; Lys11-linked chains are involved in ERAD (endoplasmic reticulum-associated degradation) and in cell-cycle control; Lys29-linked chains are involved in lysosomal degradation and kinase modification; Lys33-linked chains are involved in kinase modification; Lys48-linked chains are involved in protein degradation via the proteasome; Lys63-linked chains are involved in endocytosis, DNA-repair and NF-κB activation. Linear polymer chains formed via attachment by the Met lead to kinase activation and cell signaling in NF-κB pathway. Unanchored free polyubiquitin chains also can be generated and have distinct roles, such as in activation of protein kinases and in signaling. Ubiquitination is a reversible modification. Ubiquitin removing from the protein occurs due to activity of deubiquitinating enzymes (DUBs) [8-12]. Two main classes of DUBs, cysteine proteases and metalloproteases, are involved in protein deubiquitination. Cysteine DUB protease have a Cys in active center and include 4 main superfamilies: ubiquitin-specific proteases (USPs), ubiquitin C-terminal hydrolases (UCHs), Machado-Josephin domain proteases (MJDs), and ovarian tumour proteases (OTU). Metalloproteases have a Zn in active center and belong to MPN+/JAMM superfamily. The increased activity of DUBs may cause a problem in isolation and identification of ubiquitinated proteins from whole cellular extracts. Ubiquitin modification of target proteins is recognized by a variety of ubiquitin receptors, that carry one or several ubiquitin-binding domains (UBDs) [7, 13]. More that 20 different families of UBDs have been described. They have different structures but the same feature of noncovalently binding to ubiquitin.

Ubiquitin mutants Using different ubiquitin mutants to study the type of ubiquitin chains Mutations at the ubiquitin Lys that can participate in the chain formation are useful tool for in vitro and in vivo studies of the type of the polyubiquitin chains. These ubiquitin mutants are fully functional for activation and thiolester formation by E1, E2 and E3 enzymes, since the C-terminal residues are intact. Point-Lys ubiquitin mutants. Mutation of the one of the 7 Lys to Arg residues in ubiquitin renders ubiquitin unable to form the chains via that specific Lys with other ubiquitin 3

molecules [14]. However, they can still be linked to the target proteins and to each other via the remaining ubiquitin Lys residues that have not been mutated. The shortage of the polyubiquitin chain or complete disappearance of polyubiquitinated product will indicate that this particular Lys is involved in the chain formation. Single-Lys ubiquitin mutants – another rigorous tool to define the type of polyubiquitin chain. In these mutants all Lys, except the particular one, are mutated to Arg. In this case only the chain formed by the remaining non-mutated ubiquitin will be observed. Single-Lys mutants of ubiquitin can also be used in binding studies to determine affinities of the ubiquitin receptors to the different polyubiquitinated chains. Aside from the more commonly used forms of linkage identification detailed above, it is also possible to determine ubiquitin chain type through the use of DUBs that specifically cleave ubiquitin moieties from target proteins. Due to structural disparities between multiubiquitin chains of differing linkage, DUBs show binding-site specificity for particular linkages [15, 16]. For example, the DUB OTU7B shows specificity toward K11-linked chains [17, 18]. This specificity could be exploited to determine chain linkage in vitro. Non-Lys ubiquitin mutants. There are also non-Lys residues that are critical for ubiquitin function and structure. The ubiquitin surface, that is mainly polar, has a large hydrophobic patch formed by the Leu8, Ile44 and Val70 residues. This hydrophobic patch is necessary and important for chain recognition by UBDs [13]. Thus, the mutation of these residues (usually Ile44) that affects the structure of this hydrophobic surface, is used to study the recognition of the ubiquitinated proteins by the specific receptors. Another 2 additional important hydrophobic areas are formed by Ile36, Leu71, Leu73 and Gln2, Phe4, Thr12 [7, 19]. For in vitro ubiquitination studies commercially available synthetic ubiquitin mutants can be used. For in vivo studies the ubiquitin mutants have to be expressed from the plasmids. In the last case the ubiquitin derivatives may be tagged with affinity tag such as His6-tag and the method of affinity isolation can be used.

Identification of ubiquitinated targets in vivo Strategies for identification of ubiquitinated proteins The main strategy for identification of ubiquitinated proteins includes first isolation of the pull of ubiquitinated proteins from cellular extracts and then identification of these proteins. Isolation is performed by different pull down techniques that consist mainly in either using 4

tagged ubiquitin derivatives and affinity against the tag, or using the affinities that can recognize endogenous untagged ubiquitin. Isolated ubiquitinated targets can be identified with specific antibodies or mass spectrometry. Tagged ubiquitin allows to fish the ubiquitinated targets from the total extracts using affinity chromatography. Many differently tagged derivatives of ubiquitin have been described [20]. Some of them, such as biotinylated ubiquitin is produced in vitro, some, such as GST-, Myc-, His-, Flag-, or HA-tagged ubiquitin are produced in vivo. For in vitro assays many of the tagged versions of ubiquitin are commercially available from BIOMOL, BostonBiochem, Calibrochem, Enzo Life Science, etc. The main strategies for identification of ubiquitinated proteins are overview below and listed in the Table 1.

Stabilization of the ubiquitin conjugates in crude extracts There are several strategies to increase the yield of the ubiquitin conjugates that can be used. 1) Decreasing the level of endogenous non-tagged ubiquitin that can compete with the tagged ubiquitin. This strategy was used in yeast. In the baker’s yeast the strains SUB280 and SUB288 were created in the laboratory of D. Finley [1, 21, 22]. In these strains all 4 ubiquitin genes were deleted. Essential ribosomal proteins were expressed without ubiquitin fusion. The cell viability was maintained by a plasmid encoded the UBI4 gene with either the LYS2 (SUB280) or URA3 (SUB288) markers. SUB280 can be transformed with a plasmid containing tagged ubiquitin with URA3 marker for positive selection, followed by negative selection with α-amino-adapic acid against UBI4-LYS2 plasmid [23]. SUB288 can be transformed with a plasmid expressing tagged ubiquitin with LYS2 marker for positive selection, followed by negative selection with 5-fluroortic acid against UBI4-URA3 plasmid [24]. Both these strategies allow to produce cells expressing only tagged version of ubiquitin [22, 25, 26]. 2) Another strategy is to significantly increase the level of production of the tagged ubiquitin compare to endogenous non-tagged protein. Using of the strong inducible promoters allow to increase the expression of the desirable derivative of ubiquitin in yeast. One of the favorite promoters is a yeast metallothionein promoter, CUP1. This promoter is activated by Cu2+ ions and is widely used in expression systems [27-29]. Typical concentration of 100 μM of CuSO4 in the yeast media induced very high expression of the gene of interest. 3) Using the proteasome inhibitors or conditional proteasome mutants to increase the level of ubiquitinated proteins. Many of the proteasomal inhibitors have been described [30] and 5

plenty of them are commercially available. MG-132 (carbobenzoxyl-leucinyl-leucinylleucinal) belongs to the group of peptide aldehydes - the first proteasome inhibitors to be developed. They are still the most widely used, largely due to their low cost. For mammalian cell cultures treatment with 25 μM of MG-132 for 4 h inhibits the proteasome. For yeast cells using of MG-132 requires the special yeast mutant such as erg6∆ or pdr5∆ that increase the cell wall permeability for the drags and increase its uptake [31-33]. Treatment of the yeast cells with 30-100 μM of MG-132 for 0.5-2 h inhibits the proteasome. Mutants of the proteasome (like the yeast pre1-1, pre4-1, cim3-1, etc) can also increase the yield of the ubiquitinated proteins [33-35]. 4) Using the DUBs inhibitors. One of the big problems for identification of the ubiquitinated proteins in cellular lysates is a high activity of DUBs [36]. Plenty of DUBs inhibitors are described and available. Some of them are cell-permeable, while some are not and can be used only in cell extracts. N-ethylmaleimide (NEM) is a wide used non cell-permeable inhibitor of cysteine DUBs.

Identification of ubiquitinated proteins in vivo by His-ubiquitin pull down For this type of analysis N-terminal hexa-Histidine (His6) tagged ubiquitin is used (Fig. 3). Incorporation of His6-ubiquitin into ubiquitinated proteins allows purifying them by Nichelate affinity chromatography from yeast and mammalian cell cultures [37-42]. This basis for affinity purification is known as immobilized metal affinity chromatography (IMAC). Metal divalent ions (e.g., Ni, Co, Cu, Fe) function as ligands for binding and purification of biomolecules of interest. The chelators most commonly used as ligands for IMAC are nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA). Once IDA- or NTA- resin is prepared, it can be "loaded" with the desired metal. Ni is one of the favorite metals and the resulting IMAC is usually called Ni-chelate affinity chromatography. The purified pool of ubiquitinated proteins can be analyzed for the presence of the particular protein by western blot with specific antibodies. The main advantage of this strategy is the possibility to prepare the cell lysate in strong denaturing conditions that limits DUBs activities. Procedure. Transform the yeast cells with His6-ubiquitin plasmid. Grow 100 ml of the cell culture in the presence of 100 μM of CuSO4 in the media. Collect the cells at OD600 of 1.0 by centrifugation at 3000 g for 3 min. Wash the pellet with 1 ml of H2O and transfer to the new tube. Centrifuge at 3000 g for 3 min and keep the pellets at -20°C if it is necessary.

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Weight the pellets and resuspend them till final concentration of 50-100 mg/ml in G-buffer (100 mM NaPi pH 8.0, 10mM Tris-HCl pH 8.0, 6 M guanidium-HCl, 0.1 % triton X-100). Optional: 5 mM of imidazole can be added to the buffer in order to reduce unspecific binding. Take 1 ml of the cell suspension. Add 600 μl of glass beads and disrupt with beat beater for 15 min at room temperature (RT). Transfer the liquid phase into the new tube. Centrifuge at 16000 g for 10 min at RT. Keep the supernatants. To analyze the proteins in total extracts (TE) by SDS-PAGE guanidium-HCl should be removed. For this dilute 20 μl of the supernatants in 1.2 ml of water and incubate with 10 μl of 50% Strataclean resin (Stratagene) for 20 min at RT with shaking. Centrifuge samples at 16000 g for 1 min, discard the supernatants and elute the proteins from the resin in 50 μl of Laemmli SDS sample buffer (SB). Load 3-10 μl of TE on the gel and analyze by western blot with the relevant antibodies. For Ni-pull down incubate 700 μl of the supernatants with 30 μl of 100 % Ni-NTA agarose (Qiagen) or with Protino-Ni-IDA resin (Machinery Nagel) for 2 h at RT with mild rotation. Wash the resin 3 times with 0.5 ml of U-buffer (100 mM NaPi pH 6.8, 10 mM Tris-HCl, 8 M urea, 0.1 % Triton X-100). Elute His6-ubiquitinated proteins with 50 μl of SB. Load 10-20 μl of Ni-eluates on the gel and analyze by western blot with the relevant antibodies. The example of the Ni-His-pull down of ubiquitinated proteins is shown of Fig. 4. In this study the small ribosomal protein Rps7A was identified as a ubiquitinated protein those ubiquitination was increased at glucose depletion from the media [43]. The ubiquitinated forms of Rps7A were detectable even before Ni-His-pull down in total extracts, and they were strongly increased after pull down. However very often only a small portion of protein is ubiquitinated and the ubiquitinated form can be detected only after Ni-His-pull down.

Identification of ubiquitinated proteins in vivo by immunoprecipitation. Epitope tags such as Myc-, HA-, Flag-, etc., on the molecule of ubiquitin are widely used. These ubiquitin derivatives are usually expressed from the episomes. Proteins modified with epitope tagged ubiquitin can be immunoprecipitated from cellular lysates using specific monoclonal antibodies against the tag. Endogenous ubiquitin can be also purified by immunoprecipitation using antibodies against ubiquitin. Many of anti-ubiquitin antibodies have been produced and available on the market. Several of them may recognize ubiquitin polymers in a linkage-dependent manner. However, due to the strong conservation of ubiquitin, it is difficult to obtain high-affinity anti-ubiquitin antibodies. One of the most 7

frequently used anti-ubiquitin antibodies is the monoclonal antibody FK2. FK2 detects both mono- and polyubiquitin, but not free ubiquitin. Unfortunately, immunoprecipitation is not compatible with fully denaturing conditions such as urea or guanidium-HCl used in case of IMAC. Lysates can be prepared in SDS-containing buffers to inactivate proteases, but need to be diluted in milder buffers to avoid the denaturation of the antibodies [44, 45]. Nevertheless, purified fractions are relatively pure and are used for the proteomic analysis [45].

Identification of ubiquitinated proteins in vivo with ubiquitin-specific affinity resin Using of the His-tagged ubiquitin and denaturing conditions can significantly decrease the ubiquitin chain removal by DUBs. However this approach, which was initially described for yeast, ideally requires the replacement of all endogenous ubiquitin sources. This is impossible to do in mammalian cell lines. The alternative method is using the ubiquitin-specific affinity resins (Fig. 5). These types of resin contain UBD mobilized on the beads. UBD-pull down allows to isolate native ubiquitinated proteins from the crude extracts without using of tagged exogenous ubiquitin [46]. Different types of UBD have been used. They can specifically recognize polyubiquitinated proteins like UBA (ubiquitin-associated) domains of Dsk2 or Rad23 [47], or they can have more specificity to monoubiquitinated targets, like CUE (Cue1phomologous) [48] or UIM (ubiquitin interacting motif) domains [49-51]. Several UBD have preference to the specific polyubiquitinated chains. For example, UBA domain of Dsk2 has more affinity to K48 chains and less to K63 chains [52] and may be used to isolate proteins with these particular modifications. UBD can be expressed and purified from bacteria and directly couplet on the resin [47]. Another possibility is to use tagged UBD and the resin with the affinity to the specific tag. UBD fused to GST (glutathione-S-transferase) tag can be pulled down with immobilized glutathione resin [53]. Many of uncoupled UBDs can be bought from BioMol, Enzo Life Science, BostonBiochem, etc. Several UBD-couplet resins are also commercially available. For example Dsk2 UBA domain immobilized on agarose is available from Enzo Life Science and from BioMol. Ubiquitin-specific affinity resin pull down has some advantages and disadvantages. It can be used with endogenous ubiquitin, and UBD can protect polyubiquitinated proteins from activity of DUBs [54]. The main problem of this technique is low affinity of UBD for ubiquitin [55]. To increase the avidity multiple UBDs, such as MultiDsk or TUBEs, have been 8

used [54, 56]. MultiDsk has 5 UBA domains of Dsk2 fused to GST tag and His6 tag [56, 57]. MultiDsk is available from AbCam. TUBEs (tandem-repeated ubiquitin-binding entities) has 4 UBA domains either from ubiquilin 1 or from human HR23A (UBA1) fused to GST tag [54]. Also TUBEs contain His6 and SV5 tags flanking 4 UBA domains, which can facilitate the detection. TUBEs-agarose is available from LifeSensors. Both MultiDsk and TUBEs bind ubiquitinated targets with very high avidity and also efficiently protect them from deubiquitination by DUBs and from degradation by the proteasome [56, 58]. UbiQapture-Q matrix from BioMol or Enzo Life Science is another specific ubiquitin-binding affinity matrix to capture ubiquitinated proteins [59, 60]. It efficiently pulls down mono-, multi-, and polyubiquitinated targets. Bound proteins can be released in their active/native form either by cleavage of ubiquitin chains from the matrix using deubiquitinylating enzymes such as USP2, or by elution with high salt buffer.

Identification of ubiquitinated proteins in vivo by double-affinity purification Double-affinity purification of ubiquitinated targets consists in the combination of 2 different affinities. It can be the combination of two techniques described above, ubiquitin-specific affinity pull down and His-ubiquitin pull down. This approach allows select specific ubiquitinated targets, such as, for example, K48 chains, at the first step and then enrich them at the second step [47] (Fig. 6). Another example is using of 2 affinity-tagged ubiquitin, such as His- and Flag-tagged ubiquitin [61]. Combination of IMAC in denaturing conditions and antibody affinity chromatography in non-denaturing conditions significantly increase the portion of polyubiquitinated targets and decrease the amount of monomers of ubiquitin in the purifications.

Identification of the ubiquitination sites and determination of the ubiquitin chain topology by mass spectrometry Mass spectrometry (MS) is a powerful tool that allows to identify which site of the protein is ubiquitinated and also may determine the topology of the polyubiquitin chains. The principles of this technique are described in many articles and were used for different organisms [62-71]. Commonly used method includes purification of the His-tagged ubiquitinated substrate with IMAC under denaturing conditions with following separation of the purified protein by the SDS-PAGE and then in-gel digestion by trypsin. For mammalian cells recently described resins containing multiple UBDs can be used [45, 72]. The digested material is subjected to 9

MS analysis (Fig. 7A). Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids Lys or Arg, except when they are followed by Pro. Trypsin digestion cannot occur at the ubiquitinated Lys residue. Thus, there is a missing cleavage site in case of modified Lys. Furthermore, after trypsin digestion the original ubiquitin molecule is cleaved to a di-peptide GG remnant that adds a monoisotonic mass of about 114 Da to the modified Lys. This modification leads to unique mass spectrometry spectra (Fig. 7A). Anti-GG remnant (K-ɛ-GG)–specific antibody has been used in a growing number of largescale experiments [73-81]. This monoclonal antibody specifically recognizes GG remnant attached to conjugated Lys after trypsin digestion. Thus, after trypsin digestion, ubiquitination sites can be enriched by immunoprecipitation and analyzed by MS. Proteomic approach can be also used to determine the topology of the polyubiquitin chains [82]. Depending on the linkage type specific ubiquitin signature peptides are produced [63] (Fig. 7A and B).

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Figure legends Figure 1. The ubiquitination pathway.

Figure 2. A. Surface representation of ubiquitin (from [83]). All seven lysines (K6, K11, K27, K29, K33, K48, and K63) and the amino-terminus (M1), shown in blue, can be conjugated to the carboxy terminus of another ubiquitin molecule. The hydrophobic patch (L8, I44, and V70), shown in green, is recognized by several ubiquitin-binding proteins (PDB accession no. 1UBQ; [84]). B. The ubiquitin chains of the different topology. Ubiquitination may occur once resulting in monoubiquitination, several times but on different Lys of the substrate, resulting in multi-monoubiquitination, and finally, several times but on the same Lys of the substrate, resulting in polyubiquitination. Depending on which Lys is involved in the chain formation, different types of polyubiquitin chains can be produced.

Figure 3. Principles of His-ubiquitin pull down of ubiquitinated proteins from crude cellular extracts. Exogenous His6-tagged (green H6) ubiquitin (red UB) is expressed in the cell. Cells are lysed and His6-ubiquitinated proteins are purified on a Ni-resin. Ni-eluates are analyzed by western blot with the antibody against the protein of interest.

Figure 4. Analysis of substrate ubiquitination in vivo by His-ubiquitin pull down assay in S. cerevisiae. Small ribosomal protein, Rps7A, is ubiquitinated in response to glucose depletion (from [43]). Yeast cells expressing His6-ubiquitin were grown in the presence of 100 μM of CuSO4 and collected at 3 different stages of growth (OD600 0.7, 2.0 and 6.0) from high glucose concentration ([Glc]) in the medium to glucose depletion. Ubiquitinated proteins were purified on a Ni-agarose (Ni-eluates) from total extracts (TE) and analyzed by western blot. The position of Rps7A and ubiquitinated Rps7A (Rps7A-Ub) are indicated on the right. Molecular weight markers are indicated on the left in kDa.

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Figure 5. Principles of UBD pull down of ubiquitinated proteins from crude cellular extracts. Proteins are modified by endogenous ubiquitin (red UB) in the cell. Cells are lysed and ubiquitinated proteins are purified on a UBD-coupled resin. Eluted material is analyzed by western blot with the antibody against the protein of interest.

Figure 6. Principles of double affinity purification of ubiquitinated proteins from crude cellular extracts. Exogenous His6-tagged (green H6) ubiquitin (red UB) is expressed in the cell. Cells are lysed and ubiquitinated proteins are purified on a UBD-coupled resin in native conditions. Proteins are eluted in the presence of urea and affinity purified on a Ni-resin. Ni-eluates are analyzed by western blot with the antibody against the protein of interest.

Figure 7. A. Principles of identification of the ubiquitination sites and determination of the ubiquitin chain topology by mass spectrometry (from [63]). Protein of interest (in yellow) carries an affinity tag (in green) and is modified by polyubiquitin chain (in red). After affinity purification protein is separated on SDS gel and is subjected to trypsin digestion. Sites of trypsin digestion are shown in violet arrows. Missing trypsin cleavage site after ubiquitinated Lys residue is shown in red arrow. The digested material is analyzed by mass spectrometry. Peptides with missing cleavage site and with additional mass of –GG di-peptide originated from ubiquitin, can be matched with database. The topology of the polyubiquitin chains can be determined with the same approach. Depending on the linkage type specific ubiquitin signature peptides are produced [63]. B. Example of the determination of the K48 chain topology by mass spectrometry. Primary structure of S. cerevisiae ubiquitin. Lys residues are shown in red. Sites of trypsin digestion around K48 are shown in violet arrows. Missing trypsin cleavage site after ubiquitinated Lys residue is shown in red arrow. Unique K48 signature peptides with additional -GG residues appears as a result of trypsin digestion.

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Tables Table 1. Different strategies for identification of ubiquitinated proteins. method

principles

advantages

disadvantages

referenc es

His-ubiquitin pull

IMAC

down

Possible to use denaturing

Tagged ubiquitin

[37-41]

[44, 45]

conditions Protection from DUBs Strong avidity Low unspecific binding

Immunoprecipitati

affinity purification

Possible to work with

Native conditions

on of ubiquitin

with antibodies

endogenous ubiquitin

Low avidity Unspecific binding

UBD pull down

ubiquitin-specific

Possible to work with

affinity

endogenous ubiquitin

chromatography

Protection from DUBs

double affinity

combination of 2

Low unspecific binding

purification

different affinity

Low avidity

[55, 56]

Tagged ubiquitin

[47, 61]

purifications

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Figures

Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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