Side-Chain Losses in Electron Capture Dissociation Improves the Identification of Pt(II)-Binding Sites on Peptides and Proteins

摘要

Radical-mediated side chain losses from the charge-reduced Pt-binding species (such as CH_3S· or CH_3 SH from Met, SH· from Cys, CO_2 from Glu or Asp, and NH_2· from amine groups) were found to be characteristic indicators for rapid and unambiguous localization of the Pt-binding sites to certain amino acid residues. Ion mobility MS shows that Pt binds to multiple sites in substance P, generating multiple conformers, which can be partially localized by CAD. The approach was then successfully applied to interpret the top-down ECD spectrum of an inter-chain Pt-crosslinked insulin dimer, insulin+Pt(NH_3)_2+insulin (>10 kDa) and one of the cisplatin crosslinking sites on insulin dimer is unambiguously identified as B-chain Lys29. Although the distance between Lys29 and Glu21 (9.6 A) is out of the Pt-crosslinking range, it is clear that the side chains of Lys29 and Glu21 are both flexible, thus the flexible motion could bring them closer and enables the crosslinking by cisplatin.