The 5-azoniaspiro[4.4]nonyl group for improved MS peptide analysis: A novel non-fragmenting ionization tag for mass spectrometric sensitive sequencing of peptides.

A novel class of ionization tags, based on 5-azoniaspiro[4.4]nonyl (ASN+) scaffold were designed for improved analysis of peptides by electrospray tandem mass spectrometry (ESI-MS/MS). A new labeling agent, 1-{[3-oxo-3-(pentafluorophenoxy)propyl]carbamoyl}-5-azoniaspiro[4.4]nonane, was developed to react with amine and/or thiol group-containing peptides. The ionization efficiency of peptides resulting from derivatization was enhanced 10-100 fold, depending on the peptide sequence and hydrophobicity of the ionization tag. The proposed tags are completely stable during collision-induced dissociation (CID) experiments: they do not undergo unwanted fragmentation via Hofmann elimination and, more importantly, they cannot be removed by intermolecular nucleophilic attack. Moreover, CID of the derivatized peptide ions generates a dominant series of y-type fragment ions with a high sequence coverage. The proposed procedure was successfully tested on digested model proteins: ubiquitin and bovine serum albumin. We also synthesized isotopically labeled analog of 5-azoniaspiro[4.4]nonyl tag to check its applicability for comparative quantitative LC-ESI-MS analysis. The obtained results indicate the general usefulness of the 5-azoniaspiro[4.4]nonyl quaternary ammonium ionization tag for LC-ESI-MS/MS sequencing and quantification of peptides, especially for those of low abundance.