Characterization of alkali induced formation of lanthionine, trisulfides, and tetrasulfides from peptide disulfides using negative ion mass spectrometry

Abstract

Peptide disulfides are unstable under alkaline conditions, resulting in the formation of products containing lanthionine and polysulfide linkages. Electrospray ionization mass spectrometry has been used to characterize major species obtained when cyclic and acyclic peptide disulfides are exposed to alkaline media. Studies on a model cyclic peptide disulfide ͑Boc Ϫ Cys Ϫ Pro Ϫ Leu Ϫ Cys Ϫ NHMe͒ and an acyclic peptide, oxidized glutathione, bis C ( ␥ Glu -Cys -Gly -COOH), are described. Disulfide cleavage reactions are initiated by the abstraction of C ␣ H or C ␤ H protons of Cys residues, with subsequent elimination of H 2 S or H 2 S 2 . The buildup of reactive thiol species which act on intermediates containing dehydroalanine residues, rationalizes the formation of lanthionine and polysulfide products. In the case of the cyclic peptide disulfide, the formation of cyclic products is facilitated by the intramolecular nature of the Michael addition reaction of thiols to the dehydroalanine residue. Mass spectral evidence for the intermediate species is presented by using alkylation of thiol groups as a trapping method. Mass spectral fragmentation in the negative ion mode of the peptides derived from trisulfides and tetrasulfides results in isulfide bonds are widely observed in naturally occurring peptides and proteins. Considerable effort has been spent on mass spectrometry based methods for establishing the presence and assigning the location of cysteine residues involved in the formation of disulfide bridges in natural polypeptides [1][2][3][4][5][6][7][8][9][10][11][12]. The presence of disulfide bridges in peptide natural products can be established under conditions of negative ion mass spectrometry with neutral loss of H 2 S 2 serving as a diagnostic. Abstraction of the C ␣ -hydrogen results in formation of a peptide enolate at Cys residues, which can subsequently cleave to yield dehydroalanine residue with loss of H 2 S or H 2 S 2 . The use of negative ion mass spectrometry [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] for the study of disulfide containing peptide natural products has been established in an extensive series of investigations by Bowie and coworkers [30 -35] and other groups [36 -38]. During the course of recent attempts to fragment the negative ions of disulfide bridged peptides under mass spectrometric conditions, we noted that the cleavage reactions closely resemble those observed during breakage of disulfide bonds under alkaline conditions [39,40]. Under basic conditions, both ␣-and ␣ ␤-hydrogens of cysteine residues can be abstracted ␤ leading to two distinct modes of cleavage of the disulfide bonds, as noted by Parker and Kharasch in their comprehensive review of the mechanism of scission of sulfur-sulfur bonds [41]. The abstraction of an ␣-proton ␣ leads to the formation of a dehydroalanine residue and a persulfide, while abstraction of the ␤-proton results in ␤ formation of a thioaldehyde and a free thiol (cysteine). These processes are facile in both basic media ...

Topology of the disulfide bonds in the antiviral lectin scytovirin

“…The results presented here resonate with those recently published 12,13,23,24 and demonstrate that sample preparation steps adopted in the ''classical'' protein chemistry approaches, that include protein fragmentation combined with mass spectrometry and/or Edman sequencing, can lead to erroneous conclusions concerning protein structure and that detailed characterization of each step is necessary to assure that the results reflect the true protein structure. They may also help in clarifying some of the reasons for the difference between the previously reported NMR and crystal structures of SVN.…”

“…The topology of disulfides in SVN was originally assigned by matching the masses of its tryptic fragments obtained by LC-MS, 1 as well as of fragments of expressed SD1. 2,5 Starting with assignment of species ionizing as m/z ¼ 1318.6 (residues 30-43; C32-C38; SD1), m/z ¼ 1553.6 (residues 79-95; C80-C86; SD2), and m/z ¼ 3157.5 (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] to [51-59]; C7-55), the remaining two species, termed here ''cystine clusters'' of m/z ¼ 2511.0 and m/z ¼ 2719.1, each containing two disulfides, C20-C26/C32-C38 (SD1) and C68-C74/C80-C86 (SD2), were assigned by process of elimination.…”

“…1(B)]. While we have not analyzed in greater detail the fate of other fragments constituting the parent cystine clusters, work of others, 6 most recently exemplified on conversions of single-Cystine containing peptides, 12,13 points out to the complex phenomena.…”

“…At alkaline pH, cystine beta-elimination, and desulfurization with subsequent formation of alternative cross-links (such as the lanthionine thioether links) are common 8,10,11 and observable by modern mass spectrometry techniques. 12,13 However, unlike cystine beta-elimination/desulfurization, cystine interchange is more difficult to trace by current analytical approaches and has long been recognized as the confounding factor in protein structural analyses; if not controlled or minimized, artifactual assignments may ensue. 6 The presence of different species of the same protein with different topology of disulfide bridges has been reported only very rarely, and in most cases, the putative differences were later found to be artifacts of experimental procedures.…”

“…As can be seen from fragment assignments [ Fig. 2(A), Table II], a heterodimeric peptide, residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]-[51-60], containing C7-C55, has been identified (fragment 10). Its Edman degradation yielded two N-terminal sequences, including di-PTH-Cystine in Cycle 7, confirming the presence of a disulfide bridge.…”