SPITC Modification

4-Sulfophenyl isothiocyanate (SPTIC) reagent has been used to sulfonate the a-amino group of the N-termini and the iso-N-termini of singly charged Ub- isopeptides to enhance their analysis using MALDI-TOF/TOF-based mass spectrometry high-energy CID [52]. The protons from sulfonic acid groups of the sulfonation modification of these two N-termini results in Edman-type cleavage of the modification and first amino acid present at the N-terminus, which consequently facilities the fragmentation of a unique cluster of characteristic signature ions at higher m/z values. This enables the identification of a Ub-isopeptide with (i) two N-termini amino acids and (ii) the GG iso-chain. Characteristic signature fragmentation ions resulting from the sulfonation tags of the Ub-isopeptides include abundant ion signals representing the m/z of (i) the loss of a tag and the first N-terminal amino acid of the Ub-isopeptide backbone from the m/z of the singly charged Ub- isopeptide molecular ion and (ii) the loss of the second tag and the first iso-N-terminal amino acid from the iso-chain from the m/z of the singly charged Ub-isopeptide molecular ion. This indicates the presence of a glycine amino acid residue at the iso-N-terminus and thus confirms the presence of the GG iso-chain. In addition to the cluster of characteristic signature fragmentation ions, sequence-specific y-type product ion are generated, which enable further confirmation of the GG iso-chain attached to the acceptor lysine residue. The acidic nature, which the sulfonation modification imparts on the Ub-isopeptide is significant. The fragmentation mechanism of the sulfonated Ub-isopeptides under high-energy CID conditions results in the formation of b-type product and related ions that are neutralized or negative under positive-ion mode. This produces an exclusive series of y- type product ions on the CID MS/MS spectrum. This method also showed utility on three tryptic Ub-isopeptides generated from a tryptic digest of a ubiquitinated C-terminal Hsc70 interacting protein generated in vitro [53].

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