title: Disulfide bond cleavage in TEMPO-free radical initiated peptide sequencing mass spectrometry.
abstract: 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 MS(n) 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 abstraction of a hydrogen atom at C(β) by the benzyl radical is proposed to be the initial radical abstraction/transfer reaction. On the other hand, H-abstraction at C(α) is suggested to lead to C-S bond cleavage, which yields [ion ± S] fragments or the loss of •SH or •SSH.
authors.affiliation: Department of Chemistry, Sogang University, Seoul 121-742, Korea.
authors.forename: Minhee
authors.initials: M
authors.lastname: Lee
authors.forename: Younjin
authors.initials: Y
authors.lastname: Lee
authors.forename: Minhyuk
authors.initials: M
authors.lastname: Kang
authors.forename: Hyeyeon
authors.initials: H
authors.lastname: Park
authors.forename: Yeonmi
authors.initials: Y
authors.lastname: Seong
authors.forename: Bong June
authors.initials: BJ
authors.lastname: Sung
authors.forename: Bongjin
authors.initials: B
authors.lastname: Moon
authors.forename: Han Bin
authors.initials: HB
authors.lastname: Oh
mesh_terms: D053607:Adrenomedullin / Q000737:chemistry; D000595:Amino Acid Sequence; D004220:Disulfides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D005609:Free Radicals / Q000737:chemistry*; D006801:Humans; D007028:Hypothalamic Hormones / Q000737:chemistry; D008543:Melanins / Q000737:chemistry; D008969:Molecular Sequence Data; D010456:Peptides, Cyclic / Q000737:chemistry; D010907:Pituitary Hormones / Q000737:chemistry; D020539:Sequence Analysis, Protein / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D016211:Transforming Growth Factor alpha / Q000737:chemistry
chemical_list: D004220:Disulfides; D005609:Free Radicals; D007028:Hypothalamic Hormones; D008543:Melanins; D010456:Peptides, Cyclic; D010907:Pituitary Hormones; D016211:Transforming Growth Factor alpha; C056454:bactenecin; D053607:Adrenomedullin; C019463:melanin-concentrating hormone