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K-GG Antibody

Antibody-based enrichment strategies using monoclonal antibodies have been developed and effectively utilized to directly enrich for isopeptides generated from post-tryptic digestion, which contain an internal (K)-GG iso-chain with high selectivity [64, 65]. Although it is accepted in large- and global-scale

Ub-isopeptide analysis, and site mapping this (K)-GG iso-chain is predominantly referred to as being from a Ub-isopeptide; however, it is not unique to the Ub-isopeptide. The (K)-GG iso-chain is also present on tryptic isopeptides that have been derived from substrate proteins that have undergone PTM by two other ubiquitin-like PTMs: NEDDylation or ISG15ylation. However, with biological expertise and methodologies, it has been possible to determine the regulatory state of these two PTMs under certain conditions [68, 69] and subsequently select an appropriate biological technique to distinguish them [65]. Although the use of these biological techniques is governed by their complexity of implementation and availability, it is generally assumed that the majority of isopeptides containing a (K)-GG iso-chain identified and/or quantified are predominantly from the Ub-isopeptides with an understanding and appreciation that a percentage of these isopeptides are derived from NEDDylation or ISG15ylation. Aside from this caveat of (K)-GG isopeptide iso-chain selectivity, an additional caveat to the K-e-GG antibodies is that they demonstrate a preference for certain amino acids adjacent to the lysine residue bearing the GG iso-chain. For example, the clone GX41 monoclonal antibody [64] demonstrates a preference toward amino acid residues including leucine, isoleucine, and tyrosine, whereas the rabbit monoclonal antibody [65] demonstrates a degree of preference for aspartic and glutamic acid residues [70]. Enrichment of Ub-isopeptides post-tryptic digestion in combination with subsequent LC-MS/MS and bioinformatic analyses enables a substantial advantage in that it has greatly increased the discovery number toward thousands to tens of thousands of ubiquitination sites on hundreds and thousands of proteins in identification and quantification studies on mammalian cell and tissue samples on a global scale [70-76]. By comparison, in terms of numbers, these are substantial when considering a study conducted on mammalian cell samples using a double-affinity-tagged ubiquitin protein-level enrichment in combination with LC-MS/MS analysis and bioinformatic analyses resulted in the identification of 753 ubiquitination sites on 471 proteins [77]. In addition, by further comparison, the first large-scale ubiquitination site-mapping study conducted on a yeast sample using an affinity-tagged ubiquitin protein-level enrichment strategy in combination with LC-MS/MS resulted in the identification of 110 ubiquitination sites on 72 ubiquitinated protein conjugates from a total of 1075 proteins identified [78]. The vast increase in the numbers of ubiquitination sites that have been identified due to this type of antibody enrichment strategy render it a powerful tool in the proteomic workflow of Ub-isopeptide analysis. To further enhance Ub-analysis, modification of isopeptides using approaches such as the RUbI [54] or MEDUSA [60] approach could be implemented postantibody enrichment, thereby enabling comprehensive structural elucidation of the GG iso-chains. However, this would be dependent on the development of appropriate bioinformatic software to accommodate global analysis LC- MS/MS data.

Applying COFRADIC as an enrichment strategy to analyze Ub-isopeptides is the most recent approach to have emerged in effective Ub-G-isopeptide enrichment [66]. This enrichment strategy requires two stages: (i) sample preparation and (ii) application of COFRADIC.

i) The sample preparation stage involves (i) first, blocking all free primary protein amino groups via acetylation with NHS-acetate. (ii) Second, the use of the catalytic core of a USP2 DUB enzyme - USP2cc. USP2cc is used to deubiquitinate ubiquitinated proteins by cleaving the isopeptide bond formed between the C-terminus of the ubiquitin protein and the e-amino group of the side chain of a target lysine residue from a protein or the a- amino group of an N-terminal target lysine residue from a protein (formed from N-terminal ubiquitination of protein targets), resulting in the reintroduction of the primary protein amino groups of these target lysines. (iii) Third, selective chemical modification of these reintroduced primary amino groups via acylation with Gly-Boc-OSu to introduce a Gly-Boc group (G-Boc). (d) Tryptic digestion of the sample results in isopeptides with an internal lysine bearing a G-Boc iso-chain or peptides with N-terminal lysine bearing a G-Boc at its N-terminus.

ii) Applying COFRADIC involves (i) the tryptic sample being run on a primary RP-HPLC separation where fractions are collected and pooled; (ii) typically, a pooled fraction is then treated with 10% TFA to cleave off the Boc group from the G-Boc modification, resulting in a chemical change to the structure of the Ub-G-isopeptide or N-terminal Ub-G-peptide, specifically evoking a hydrophilic retention time shift of these Ub-G-isopeptides or N-terminal Ub-G-peptides, which is observed in (iii). (iii) The samples are then subjected to a secondary RP-HPLC separation under identical conditions to the primary RP-HPLC separation. The hydrophilic retention time shift that had been evoked from the cleavage of the Boc group in (ii) is observed during the secondary RP-HPLC run with the Ub-G-isopeptides and N-terminal Ub-G-peptides chromatographically separating from peptides, which did not undergo a hydrophilic retention time shift. This enables fractions of enriched Ub-G-isopeptides and N-terminal Ub-G-peptides to be collected and prepared for subsequent LC-MS/MS analysis. A detailed scheme of this COFRADIC-based enrichment strategy is depicted in Figure 6.9. (Note: Ub-G-isopeptides and N-terminal Ub-G-peptides are referred to in the reproduced Figure 6.9 as Gly-BOC-peptides.)

This COFRADIC-based enrichment strategy was effective in enabling the identification of 7504 ubiquitinated lysines on 3338 proteins along with 9 ubiquitinated protein N-termini from human Jurkat cells [66]. It was reported that the number of ubiquitinated lysines identified was 43% higher than those

An illustration of the COFRADIC workflow for the analysis of ubiquitination. Source

Figure 6.9 An illustration of the COFRADIC workflow for the analysis of ubiquitination. Source: Stes 2014 [66]. Reproduced with permission of American Chemical Society.

reported in studies using epitope-tagged ubiquitin [77] and the (K)-GG iso-chain-specific antibodies [74]. Furthermore, COFRADIC enabled the identification of sites of N-terminal ubiquitination, which would not have been possible with the (K)-GG iso-chain-specific antibodies due to their specificity for the internal (K)-GG iso-chain. A caveat to this enrichment strategy is that the acetylation reaction was determined to reach a completion of 95%, ultimately resulting in the misassignment of a proportion of the ubiquitination sites; in this case, it was 6.7% (at the isopeptide/peptide level) [66]. The authors have indicated that this strategy can be applied to quantitative analysis using stable isotopic labeling.

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