Enrichment and Global Analysis of Isopeptides
Overview of Enrichment Approaches
The dynamically reversible PTM of mammalian substrate proteins by wild-type mammalian ubiquitination and SUMOylation occurs at sub-stoichiometric levels. These sub-stoichiometric levels greatly limit the analytical coverage of putative Ub- and SUMO-isopeptides in a background of thousands of more abundant peptides generated postproteolytic digestion of complex biological samples. This subsequently limits the number of putative modification sites that can be analyzed and uniquely identified on a large or global scale by MS analyses. In order to circumvent this, enrichment strategies have been developed and incorporated into Ub- and SUMO-specific proteomic work flows with the ultimate aim of enriching for Ub- and SUMO-isopeptides. These enrichment strategies are implemented at the following stages: the pre-analytical stage:
i) protein level; this level focuses on the isolation and purification of Ub and/ or SUMO proteins for subsequent analytical analyses, which are of interest to this book or alternative types of analyses but beyond the scope of this chapter;
ii) isopeptide level; this level focuses on the enrichment of isopeptides containing an internal lysine (K) bearing a GG iso-chain ((K)-GG iso-chain), the analytical stage;
iii) chromatographic level; this level focuses on the enrichment of generated Ub-isopeptides bearing a single G amino acid, through the use of automated chromatography using the elegant application of combined/ractional diagonal chromatography (COFRADIC).
Of course, these isopeptides are in contrast to the classical Ub-isopeptides that bear the classical GG iso-chain from direct tryptic of digestion of ubiquitinated proteins. For simplicity, we refer to these enrichment-approach-generated isopeptides as Ub-G-isopeptides or N-terminal Ub-G-peptides (if an N-terminal G-Boc modification is present). This enrichment strategy has been demonstrated for the effective analysis of Ub-G-isopeptides and enhanced analysis of N-terminal Ub-G-isopeptides, with a view to being applied to the analysis enrichment-approach-generated SUMO-isopeptides.
Inclusion of enrichment strategies for wild-type ubiquitinated proteins or Ub-isopeptides at the
i) protein level involves the use of (a) affinity- or epitope-tagged ubiquitin proteins , ubiquitin-binding domain (UBD) proteins , and tandem ubiquitin-binding entities (TUBES) proteins , resulting in subsequent purification of ubiquitinated and polyubiquitinated proteins; (b) anti- ubiquitin antibodies that have the capability to recognize ubiquitinated and polyubiquitinated proteins [48, 61] although additional PTMs involved in possible cross-talk with ubiquitination such as SUMO(2/3)ylation can also be recognized .
ii) isopeptide-level enrichment involves the use of monoclonal antibodies that recognize and are highly specific to the (K)-GG iso-chain [64, 65], which is internally present on isopeptides that have been generated post- tryptically from substrate proteins that have been post-translationally modified by ubiquitin, Nedd, or ISG15.
iii) analytical analysis-level enrichment involves the use of automated reversed-phase high-performance liquid chromatography (RP-HPLC) using COFRADIC. The application of COFRADIC enables the enrichment of Ub-G-isopeptides and N-terminal Ub-G-peptides by utilizing changes in their hydrophilicity in response to a selective change in their chemical structure .
A combination of enrichment strategies, known as dual-enrichment strategies, using types of TUBES known as Trypsin Resistant-TUBES (TR-TUBES) at the protein level and the monoclonal K-e-GG antibody at the isopeptide level have demonstrated an enhancement in the analysis of Ub-isopeptides related to ligase substrates and activity . Ultimately each level of enrichment that is incorporated into the proteomic workflows is conducted with a view to maximize the analytical and bioinformatic analyses of protein ubiquitination or SUMOylation. Each of the enrichment strategies has its advantages and disadvantages, although the progression of the enrichment strategies over time has resulted in vastly improving the depth of our biological knowledge of these PTMs. A few selected examples from these enrichment strategies that have been developed at the isopeptide level and analytical level for Ub- isopeptides and the protein level for protein SUMOylation and isopeptide level for SUMO-isopeptides are discussed further.