Disulfide bond cleavage in TEMPO-free radical initiated peptide sequencing mass spectrometry

• Published in: Journal of mass spectrometry. Vol. 46; no. 8; pp. 830 - 839
• Main Authors: Lee, Minhee, Lee, Younjin, Kang, Minhyuk, Park, Hyeyeon, Seong, Yeonmi, June Sung, Bong, Moon, Bongjin, Bin Oh, Han
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01.08.2011; Wiley
• Subjects: Adrenomedullin - chemistry; Amino Acid Sequence; Aminoacids, peptides. Hormones. Neuropeptides; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Bonding; Cleavage; disulfide bond cleavage; Disulfides; Disulfides - analysis; Disulfides - chemistry; Disulfides - metabolism; Fragmentation; Fragments; free radical initiated peptide sequencing; Free Radicals - chemistry; Fundamental and applied biological sciences. Psychology; Humans; Hypothalamic Hormones - chemistry; Mass spectrometry; Melanins - chemistry; Molecular Sequence Data; odd-electron tandem mass spectrometry; peptide sequencing; Peptides; Peptides, Cyclic - chemistry; Pituitary Hormones - chemistry; Proteins; Radicals; Sequence Analysis, Protein - methods; Tandem Mass Spectrometry - methods; TEMPO; Transforming Growth Factor alpha - chemistry; Tandem mass spectrometry; Nitroxyl; Reaction path; Fragmentation pattern; Primary structure; free radical initiated peptide sequencing; Peptide map; disulfide bond cleavage; peptide sequencing; Disulfide bond; Organic free radical; Cleavage; odd-electron tandem mass spectrometry; Gas phase; TEMPO; Mass spectrometry; Aminoacid sequence; Structural analysis
• ISSN: 1076-5174; 1096-9888; 1096-9888
• DOI: 10.1002/jms.1955

The gas‐phase free radical initiated peptide sequencing (FRIPS) fragmentation behavior of o‐TEMPO‐Bz‐conjugated peptides with an intra‐ and intermolecular disulfide bond was investigated using MSn tandem mass spectrometry experiments. Investigated peptides included four peptides with an intramolecular cyclic disulfide bond, Bactenecin (RLCRIVVIRVCR), TGF‐α (CHSGYVGVRC), MCH (DFDMLRCMLGRVFRPCWQY) and Adrenomedullin (16–31) (CRFGTCTVQKLAHQIY), and two peptides with an intermolecular disulfide bond. Collisional activation of the benzyl radical conjugated peptide cation, which was generated through the release of a TEMPO radical from o‐TEMPO‐Bz‐conjugated peptides upon initial collisional activation, produced a large number of peptide backbone fragments in which the SS or CS bond was readily cleaved. The observed peptide backbone fragments included a‐, c‐, x‐ or z‐types, which indicates that the radical‐driven peptide fragmentation mechanism plays an important role in TEMPO‐FRIPS mass spectrometry. FRIPS application of the linearly linked disulfide peptides further showed that the SS or CS bond was selectively and preferentially cleaved, followed by peptide backbone dissociations. In the FRIPS mass spectra, the loss of •SH or •SSH was also abundantly found. On the basis of these findings, FRIPS fragmentation pathways for peptides with a disulfide bond are proposed. For the cleavage of the SS bond, the ion of a hydrogen atom at Cβ by the benzyl radical is proposed to be the initial radical ion/transfer reaction. On the other hand, H‐ion at Cα is suggested to lead to CS bond cleavage, which yields [ion ± S] fragments or the loss of •SH or •SSH. Copyright © 2011 John Wiley & Sons, Ltd.