Ubiquitin is 8.5 kDa polypeptide consisting of 76 amino acids. It is highly conserved among eukaryotes and can be covalently attached to Lys target, either as a monomer or as a Lys-linked polymer. Conjugation of ubiquitin to intracellular proteins can alter the property of its targets in a variety of ways, from localization, activity, partner binding, to their selective degradation.

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Figure 1. The ubiquitination pathway.

A detailed protocol for the detection of linear poly-ubiquitination chains by immunoblotting has been developed and published in 2015 [5]. The work of Sasaki et al exemplifies the use of an anti-linear ubiquitin antibody for the detection of poly-ubiquitin chains in the regulatory subunit, NEMO/IKKγ, of the IKK kinase, the core element of the NF-κB cascade [5]. Kabayama H et al detected poly-ubiquitin chains in immunocytochemistry with a monoclonal antibody [6].

In Saccharomyces cerevisiae , ubiquitin is coded by 4 genes UBI1-4 [7]. 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 such as high temperature, and starvation. 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 among species. At the protein level, it is not possible to determine from which of the 4 genes ubiquitin chain is 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 ubiquitin-conjugating 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 [8]. The human genome encodes 2 E1s, about 50 E2s and more than 600 E3s [9], while yeast has 1 E1s, 13 E2s and about 100 E3s [10]. MLN4924 (Pevonedistat) inhibits enzyme NEDD8, blocks CUL4 neddylation, and consequently E3 ligase activity and in the end prevents ubiquitination [11]. Small molecule chemicals, able to bring together a protein substrate and a ubiquitin ligase inducing the ubiquitination and eventual degradation of the substrate, called PROteolysis TArgeting Chimeras (PROTACs), are explored as a new class of therapies for cancers and Alzheimer's disease [12, 13]. Photoswitchable PROTACs (PHOtochemically TArgeting Chimeras -> PHOTACs) are being developed [14]. TRIM21, an E3 ubiquitin ligase and cytosolic antibody receptor, has been exploited to degrade a target protein inside cells in combination with an exogenously introduced antibody against the target (Trim-Away) [15, 16].

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Figure 2. A. Surface representation of ubiquitin (from [1] ). 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; [2] ). B. The ubiquitin chains with different topologies. 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.

Ubiquitination may occur once, resulting in monoubiquitination; or 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 N-terminal methionine (Met1) that can be ubiquitinated. Link-specific antibodies are available against M1, K11, K27, K48 and K63 linkages, and high-affinity specific affimers have been against K6 and K33/K11 linkages [17]. 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 [18-20]. 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 the NF-κB pathway. Unanchored free polyubiquitin chains also can be generated and have distinct roles, for example, in the activation of protein kinases and in signaling.

Ubiquitination is a reversible modification. Ubiquitin removing from the protein occurs due to the activity of deubiquitinating enzymes (DUBs) [21-25]. Two main classes of DUBs, cysteine proteases and metalloproteases, are involved in protein deubiquitination. Cysteine DUB protease has a Cys in the active center and includes four 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 the 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) [20, 26]. More than 20 different families of UBDs have been described. They have different structures but the same feature of noncovalently binding to ubiquitin.

A recent comprehensive review [27] discusses the advantages and the disadvantages of the immunoblotting methods currently used to analyze protein ubiquitylation. The article includes recommendations for sample treatment in order to prevent protein deubiquitylation; sample analysis by SDS-PAGE including the choice of gel and running buffer and methods for gel-to-membrane transfer; and ways to choose the best anti-ubiquitin antibody and to enhance its performance.

Mutations at the ubiquitin Lys that can participate in the chain formation are a useful tool for in vitro and in vivo studies of the type of polyubiquitin chains. These ubiquitin mutants are fully functional for activation and thiol ester formation by E1, E2 and E3 enzymes, since the C-terminal residues are intact.

Mutation of the one of the 7 Lys to Arg residues in ubiquitin renders ubiquitin unable to form any chains via that specific Lys with other ubiquitin molecules [28]. 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 the polyubiquitinated product will indicate that this particular Lys is involved in the chain formation.

This is another rigorous tool to define the type of polyubiquitin chain. In these mutants, all Lys, except one, are mutated to Arg. In this case only the chain formed by the non-mutated ubiquitin will be observed. Single-Lys mutants of ubiquitin can also be used in binding studies to determine affinities of ubiquitin receptors to 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 [29, 30]. For example, DUB OTU7B shows specificity toward K11-linked chains [31, 32]. This specificity could be exploited to determine chain linkage in vitro .

There are also non-Lys residues critical for ubiquitin function and structure. The ubiquitin surface, mainly polar, has a large hydrophobic patch formed by Leu8, Ile44 and Val70 residues. This hydrophobic patch is necessary and important for chain recognition by UBDs [26]. Thus, a mutation of these residues (usually Ile44) that affects the structure of this hydrophobic surface, is used to study the recognition of ubiquitinated proteins by specific receptors. Two additional important hydrophobic areas are formed by Ile36, Leu71, Leu73 and Gln2, Phe4, Thr12 [20, 33].

For in vitro ubiquitination studies, commercially available synthetic ubiquitin mutants can be used. For in vivo studies ubiquitin mutants have to be expressed from plasmids. In the last case the ubiquitin derivatives may be tagged with an affinity tag such as His6-tag and affinity isolation can be used.

The main strategy for identification of ubiquitinated proteins includes the isolation of ubiquitinated proteins from cellular extracts and then the identification of these proteins (Table 1). Isolation is performed by pull-down techniques either using tagged ubiquitin derivatives, or using affinity reagents that can recognize endogenous untagged ubiquitin. Isolated ubiquitinated targets can be identified with specific antibodies or mass spectrometry.

Tagged ubiquitin allows to fish ubiquitinated targets from total extracts using affinity chromatography. Differently tagged derivatives of ubiquitin have been described [43]. Some of them, such as biotinylated ubiquitin is produced in vitro [44] ; while others such as GST-, Myc-, His-, Flag-, or HA-tagged ubiquitin, for example [45], are produced in vivo . For in vitro assays, many tagged ubiquitins are commercially available from BIOMOL, BostonBiochem, Calbiochem, Enzo Life Science, etc or labelled directly in a lab, for example, with TAMRA-maleimide [46].

Several strategies can be used to increase the yield of ubiquitin conjugates.

This strategy was used in yeast. In the baker’s yeast the strains SUB280 and SUB288 were created in the laboratory of D. Finley [7, 47, 48]. 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 encoding 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-adipic acid against UBI4-LYS2 plasmid [49]. SUB288 can be transformed with a plasmid expressing tagged ubiquitin with LYS2 marker for positive selection, followed by negative selection with 5-fluoroortic acid against UBI4-URA3 plasmid [50]. Both these strategies allow to produce cells expressing only the tagged version of ubiquitin [48, 51, 52].

Using 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 [53-55]. A typical concentration of 100 µM of CuSO₄ in the yeast media induces very high expression of a gene of interest.

A dedicated review article about proteasomal inhibitors is available. MG-132 (carbobenzoxyl-leucinyl-leucinyl-leucinal) belongs to the group of peptide aldehydes - the first proteasome inhibitors to be developed. It is still the most widely used, largely due to its low cost. For mammalian cell cultures, treatment with 25 µM of MG-132 for 4 h inhibits the proteasome. For yeast cells, application of MG-132 requires special yeast mutants such as erg6∆ or pdr5∆ that increase cell wall permeability and increase its uptake [56-58]. Treatment of 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 ubiquitinated proteins [59, 60].

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

One of the big problems for the identification of ubiquitinated proteins in cellular lysates is the high activity of DUBs [61]. Plenty of DUBs inhibitors are described and available. Some of them are cell-permeable, while others are not and can be used only in cell extracts. N-ethylmaleimide (NEM) is a widely used non-cell-permeable inhibitor of cysteine DUBs.

N-terminal hexahistidine (His6) tagged ubiquitin is used (Fig. 3). Incorporation of His6-ubiquitin into ubiquitinated proteins allows their purification by Ni-chelate affinity chromatography from yeast and mammalian cell cultures [62-67]. The purified pool of ubiquitinated proteins can be analyzed for the presence of a 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 limit DUBs activities.

Transform the yeast cells with an 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 necessary.

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.

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Figure 4. Analysis of substrate ubiquitination in vivo by His-ubiquitin pulldown assay in S. cerevisiae. Small ribosomal protein, Rps7A, is ubiquitinated in response to glucose depletion (from [3] ). 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.

For analysis of the proteins in total extracts (TE) by SDS-PAGE, guanidium-HCl should be removed. 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 the 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 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 relevant antibodies.

An example of Ni-His-pull down of ubiquitinated proteins is shown of Fig. 4. Small ribosomal protein Rps7A was identified as a ubiquitinated protein and the ubiquitination increased at glucose depletion from the media [3]. The ubiquitinated forms of Rps7A were detectable even before Ni-His-pull down in total extracts, and their levels increased after pulldown. However very often, only a small portion of protein is ubiquitinated and the ubiquitinated form can be detected only after Ni-His-pull down. Lee YR et al transduced PC3 cells and MEF cells with His-ubiquitin and Myc-PTEN expression vectors to investigate the ubiquitination of PTEM in vivo [34]. Using His-ubiquitin pulldown, immunoprecipitation and immunofluorescence, Yang et al have shown that ribosomal protein L6 interacts with histone H2A [35]. Moreover, the authors have demonstrated that protein L6 is translocated to DNA damage sites and regulates interaction between mediator of DNA damage checkpoint 1 (MDC1) and H2A histone [35].

Epitope tags such as Myc-, HA-, Flag- 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 cell lysates using specific monoclonal antibodies against the tags. Endogenous ubiquitin can be also purified by immunoprecipitation using antibodies against ubiquitin. Many ubiquitin antibodies have been produced and are available on the market. Several of them 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 frequently used anti-ubiquitin antibodies is the monoclonal antibody FK2. FK2 detects both mono- and polyubiquitin, but not free ubiquitin. Ling Q et al, for instance, immunoprecipitated Toc33-HA with an anti-HA antibody from Arabidopsis protoplast lysates and quantified its ubiquitination with an anti-ubiquitin antibody [68]. Immunoprecipitation has also been one of the methods to detect the ubiquitinated proteins in the cells expressing mutated cysteine protease deubiquitinase UCHL5 [36] and to measure the levels of Na+-K+-2Cl- cotransporter [37]. The detected proteins have included proteasome substrates and subunits. In addition, Yang et al have used immunoprecipitation to measure the amount of ubiquitin bound to p27 in multiple myeloma cells treated with S-phase kinase-related protein 2 (Skp2) inhibitor [38].

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

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 [69, 70]. Nevertheless, purified fractions are relatively pure and are used for proteomic analysis [70].

Using 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 [71]. Different types of UBD have been used. They can specifically recognize polyubiquitinated proteins like UBA (ubiquitin-associated) domains of Dsk2 or Rad23 [41], or they can have more specificity to monoubiquitinated targets, like CUE (Cue1p-homologous) [72] or UIM (ubiquitin interacting motif) domains [73-75]. Several UBD have a preference for the specific polyubiquitinated chains. For example, the UBA domain of Dsk2 has more affinity to K48 chains and less to K63 chains [76] and may be used to isolate proteins with these particular modifications.

UBD can be expressed and purified from bacteria and directly coupled on the resin [41]. 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 [77]. 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 pulls down has some advantages and disadvantages. It can be used with endogenous ubiquitin, and UBD can protect polyubiquitinated proteins from the activity of DUBs [78]. The main problem of this technique is the low affinity of UBD for ubiquitin [39]. To increase the affinity for multiple UBDs, such as MultiDsk or TUBEs, have been used [40, 79]. MultiDsk has 5 UBA domains of Dsk2 fused to a GST or a His6 tag [40, 80]. TUBEs (tandem-repeated ubiquitin-binding entities) contain 4 UBA domains either from ubiquilin 1 or from human HR23A (UBA1) also fused to a tag [78], and are commonly used to affinity purify polyubiquitinated proteins from cell extracts [78, 81]. Different types of TUBEs are now available from LifeSensors: TUBEs for protein purification (agarose-TUBEs, GST- and His6-TUBEs), TUBEs for immunohistochemistry (fluorescent TUBEs), or TUBES for Far Western (ligand) blotting (biotin -TUBEs). Both MultiDsk and TUBEs bind ubiquitinated targets with very high affinity and can protect them from deubiquitination by DUBs and from degradation by the proteasome [40, 82] efficiently. Most recently, Yoshida et al used exogenously expressed trypsin-resistant TUBEs (TR-TUBEs) to protect the polyubiquitin chains of proteins and to identify substrates of specific ubiquitin ligases [83].

UbiQapture-Q matrix from BioMol or Enzo Life Science is another specific ubiquitin-binding affinity matrix to capture ubiquitinated proteins [84, 85]. 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. A new study reports the generation of an antibody to linear polyubiquitin chain suitable for immunofluorescence [86]. The antibody to K48-linked polyubiquitin positively reacted with the cellular components of the neurons in the brains of individuals with Alzheimer’s disease.

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

A novel approach to detect ubiquitinated proteins, known as ligase-trapping, is an affinity purification method. The technique uses an E3-ligase polyubiquitin-binding domain fusion to isolate specific ubiquitinated substrates that are further analyzed by mass spectrometry or western blot analysis. A detailed protocol has been described elsewhere [87]. O’Connor et al have described the applications of Ubiquitin-Activated Interaction Traps (UBAITs), which are used to study the ubiquitin system by covalently trapping the interactions between proteins [88]. The ubiquitin moiety on the UBAIT may link to lysine side chains of the protein, which interacts with the target protein.

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 [41] (Fig. 6). Another example is the using of 2 affinity-tagged ubiquitin, such as His- and Flag-tagged ubiquitins [42]. 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 ubiquitin monomers during the purifications.

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Figure 7. A. Principles of identification of ubiquitination sites and determination of the ubiquitin chain topology by mass spectrometry (from [4] ). Protein of interest (in yellow) carries an affinity tag (in green) and is modified by a polyubiquitin chain (in red). After affinity purification, the protein is separated on an 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 the additional mass of –GG di-peptide originated from ubiquitin, can be matched with a database. The topology of polyubiquitin chains can be determined with the same approach. Depending on the linkage type, specific ubiquitin signature peptides are produced [4]. B. An 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 the additional -GG residues appear as a result of trypsin digestion.

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 [89]. The principles of this technique are described in many articles and have been used for different organisms [4, 90-98]. Commonly used methods include 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 could be used [70, 99]. The digested material is subjected to MS analysis (Fig. 7A). Trypsin cleaves peptide chains mainly at the carboxyl side of 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 monoisotopic 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 large-scale experiments [100-108]. 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.

As identification of ubiquitinated sites by MS needs trypsin digestion, a recent study has tested the ability of LysargiNase, the mirrored trypsin, to cleave ubiquitinated sites [109]. The authors have shown that LysargiNase cleaves K63-linked chain and has higher peptidase activity than trypsin and therefore is suggested to improve detection of ubiquitinated sites.

In addition, Chicooree and Griffiths have recently reported a new technique to analyze ubiquitinated proteins by MS [110]. The method includes a simple chemical labelling using Quadrupole-Time-Of-Flight (Q-TOF) mass spectrometer. Furthermore, it allowed significant enhancement of isopeptide detection and quantification in both the precursor and product spectral domains. Also, a combination of liquid chromatography with tandem MS has allowed identification and quantification of ubiquitinated proteins [111]. The described technique has helped to detect specific alterations of the peptides induced by lentiviral-mediated transduction.

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Figure 8. Schematic diagram of the PAC-proteomics technique.

Proteomic approaches can also be used to determine the topology of the polyubiquitin chains [112]. Depending on the linkage type specific ubiquitin signature peptides are produced [4] (Fig. 7A and B). A recent review by Beaudette et al [113] provides a nice overview of the MS-based proteomics techniques that are currently available for the analysis of ubiquitin and polyubiquitin chains in proteins.

The parallel adapter capture (PAC) proteomics approach has been used recently in studies that aim to identify ubiquitination sites in ligase specific substrates [114, 115]. In this approach, single or multiple tagged ligase proteins are expressed in transfected cell lines that can be subjected to diverse treatments. Immunoprecipitation, affinity purification and mass spectrometry analysis are then used to comparatively analyze samples from treated and untreated cells. Bioinformatic analysis across samples leads to the identification of known and putatively novel ubiquitin ligase substrates (Figure 8).