MS Analysis of Modified Ub- and SUMO-Isopeptides under CID Conditions

Typical tryptic cleavage C-terminal to arginine residues at position 74 and 72 on the ubiquitin iso-chain occurs and results in the generation of a Ub-isopep- tide containing an iso-chain of 2 or 4 amino acids: GG or LRGG. It has been reported that under efficient and complete digestion conditions the GG isochain is predominantly formed over the miscleaved LRGG form [48]. However, under time-controlled digestion conditions where partial digestion is used, the LRGG form is prevalent [49]. As we have discussed, complex fragmentation patterns and generation of a number of large product ions that can arise from the full-length SUMO iso-chains under CID analysis of isopeptides render MS/ MS spectra difficult to decipher and interpret with high confidence. Analysis of Ub-isopeptides bearing the GG iso-chain and their subsequent MS/MS spectra has historically suffered from issues with false-positive identifications by bioinformatic software algorithms due to (i) misassignment of a flanking asparagine amino acid residue and alkylation of two cysteine residues, which are isobaric to the m/z of the GG iso-chain and (ii) general difficulty in interpreting MS/MS spectra manually due to the GG iso-chain of the Ub modification providing limited informative iso-chain product ions. Approaches using ETD with ion- trap-based instruments [50] and ECD on FT-ICR-based mass spectrometry [51] demonstrated more confident assignment of Ub-isopeptides due to the preservation of the GG iso-chain on the backbone of product ions related to the specific site of modification, enabled by the non-ergodic nature of the fragmentation process. However, there was still a requirement for the generation of GG or LRGG iso-chain Ub-modification-specific product ions to improve CID analysis of Ub-isopeptides. An approach developed to assess this requirement for the LRGG iso-chain modification-specific product ions involved the use of QTOF-based ESI-MS/MS and LC-nESI-MS/MS with low-energy CID analysis. It was used to analyze a synthetic model Ub-isopeptide comprising a calmodulin-related amino acid backbone. This Ub-isopeptide had been subjected to a time-controlled partial-digestion strategy using trypsin, which facilitated the generation of a Ub iso-chain containing 4 amino acids: LRGG [49]. The CID MS/MS spectrum of the Ub-isopeptide containing the LRGG iso-chain displayed predominantly y-type product ions from its backbone and two b'-type product ions from its iso-chain; b2' (LR) and b4' (GG), in addition to an internal ion, LRGGK-28 Da. The combination of these three ions indicated diagnostic confirmation of the LRGG iso-chain although their robust diagnostic utility was not assessed beyond the model peptide stage. More effective strategies that have been developed focus on using N-terminal-specific chemical derivatiza- tion, which selectively modify the a-amino groups of N-terminal and iso-N-terminal groups, along with the e-amino groups of lysine side chains. The modification of the N-terminus and iso-N-terminus imparts favorable fragmentation behavior on Ub-isopeptides (and atypically derived SUMO(2)/ (3)-isopeptides with TGG and QTGG iso-chains) under CID conditions enabling the generation of (i) characteristic diagnostic product ions from the Ub-isopeptide backbone, which indicate the site of the specific GG iso-chain Ub modification or (ii) diagnostic ions specific to the GG iso-chain Ub- modification, thus enabling complete identification of the GG iso-chain and the specific site of modification on the Ub-isopeptide backbone.

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