Site specific sequestering and stabilization of charge in peptides by supramolecular adduct formation with 18-crown-6 ether by way of electrospray ionization

Abstract

The binding of 18-crown-6-ether (18C6) to biologically relevant groups is studied by way of electrospray ionization (ESI) quadrupole ion-trap mass spectrometry. Isolated lysine is a special case in which the 18C6 attaches to the N-terminus, deriving stability from the resulting salt-bridge structure. In a peptide, 18C6 forms a stable supramolecular adduct with lysine by way of specific hydrogen bonding with the primary protonated amine on the side chain. 18C6 will bind to adjacent lysines, forming multiply charged complexes. For example, the most intense peak in the spectrum of a mixture of 18C6 and tetralysine is the quadruply charged peptide with four crown ethers attached. Competitive binding of 18C6 by other basic sites such as the n-terminus, histidine, and arginine limits the utility of this technique for the molecular recognition of lysine. Adducts of 18C6 with proteins such as cytochrome-c (CytC) and bovine pancreatic trypsin inhibitor (BPTI) reveal structural information relevant to the accessibility of potential binding sites. Adduct formation with 18C6 appears to increase the surface activity of peptides in a charged droplet and inhibits deprotonation during ion desolvation in the ESI process. These effects increase both the primary charge state and ion abundance of the analyte. Sequestering and stabilization of charge provided by 18C6 allows for the detection of species that are normally difficult to observe using ESI, such as glycine. The relationship between the observed behaviors of 18C6 adducts and the proposed ion evaporation mechanism of ESI is discussed.

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