A novel salt bridge mechanism highlights the need for nonmobile proton conditions to promote disulfide bond cleavage in protonated peptides under low-energy collisional activation

Evidence for photo-induced radical disulfide bond scrambling in the gas phase during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is described. The phenomenon was observed during the analysis of tryptic peptides from insulin and was confirmed in the determination of disulfide bonds in the rhamnose-binding lectin SEL24K from the Chinook salmon Oncorhynchus tshawytscha. A possible mechanism for this surprising scrambling is proposed. Despite this finding, the disulfide bond pattern in SEL24K was assigned unambiguously by a multi-enzyme digestion strategy in combination with MALDI mass spectrometry. The pattern was found to be symmetrical in the tandem repeat sequence of SEL24K. To the best of our knowledge, this is the first report of disulfide bond scrambling in the gas phase during MALDI-MS analysis. This observation has important ramifications for unambiguous assignment of disulfide bonds. (J Am Soc

Section: Gas-phase Scrambling Of Disulfide Bondsmentioning

confidence: 97%

“…Fragmentation across the S-S-bond (homolytic and heterolytic) provides evidence for the structure [12,13]. Among other feasible fragmentation mechanisms, one paper has reported evidence for disulfide scrambling in the gas phase during CAD [14].…”

mentioning

confidence: 99%

Gas-phase scrambling of disulfide bonds during matrix-assisted laser desorption/ionization mass spectrometry analysis

Zhao¹,

Almaraz

Fan

et al. 2009

“…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.…”

mentioning

confidence: 99%

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

Thakur

Balaram

2009

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 ...

“…Several groups have focused on the mass spectrometric analysis of peptides containing intact disulfide bonds using both positive and negative ions for gas-phase fragmentation [6 -12]. In the case of positively charged peptide ions, disulfide bond fragmentation occurs with much lower efficiency than cleavage of backbone peptide bonds, under conditions of collision induced dissociation with non-mobile protons suggested to play an important role [11]. Restricted proton mobility along the polypeptide backbone has also been implicated in cases of disulfide cleavages in polypeptides, which have been cationized using gold (I) [7].…”

mentioning

confidence: 99%

Fragmentation of peptide disulfides under conditions of negative ion mass spectrometry: Studies of oxidized glutathione and contryphan

Thakur

Balaram

2008

The fragmentation of positive and negative ions of peptide disulfides under mass spectrometric conditions yields distinctly different product ion distributions. A negative ion upon collision induced dissociation yields intense product ions, which correspond to cleavage at the disulfide linkage. The complete assignment of the product ions obtained upon fragmentation of oxidized glutathione in an ion trap is presented. The cleavage at the disulfide site is mediated by abstraction of C ␣ H and C ␤ p H protons resulting in product ions derived by neutral loss of H 2 S 2 and H 2 S. The formation of peptide thioaldehydes and persulfides at the cysteine sites is established. Dehydroalanine formation at the Cys residue is predominant. The case of a contryphan, a cyclic peptide disulfide derived from Conus snail venom, illustrates the utility of negative ion mass spectrometry in disulfide identification. M ass spectrometric characterization of disulfide bonded peptides is generally achieved by the reduction of the S-S bond followed by alkylation [1][2][3][4] or by oxidation to sulfonic acid derivatives [5]. Subsequent gas-phase fragmentation results in readily identifiable backbone cleavage products. Several groups have focused on the mass spectrometric analysis of peptides containing intact disulfide bonds using both positive and negative ions for gas-phase fragmentation [6 -12]. In the case of positively charged peptide ions, disulfide bond fragmentation occurs with much lower efficiency than cleavage of backbone peptide bonds, under conditions of collision induced dissociation with non-mobile protons suggested to play an important role [11]. Restricted proton mobility along the polypeptide backbone has also been implicated in cases of disulfide cleavages in polypeptides, which have been cationized using gold (I) [7]. The use of metal ions as gas-phase disulfide cleavage agents has also been investigated [12]. In general, only limited information regarding structure can be derived from intact disulfide peptides in the positive ion mode. In contrast, several recent studies have emphasized the utility of negative ion mass spectrometry [7,10,[13][14][15][16][17][18]. Specifically, Bowie and coworkers have presented assignments of product ion spectra obtained by negative ion electrospray mass spectrometry of peptides containing both intra and inter molecular disulfide bond [10,18]. The present study complements the work of Bowie and coworkers and further provides mechanistic details by examining second generation product ions by using ion trap mass spectrometry.In the structural analysis of peptides containing intact disulfides, under negative ion conditions, proton abstraction from the C ␣ H and C ␤ H positions of the Cys residue results in diverse fragmentation reactions, leading to cleavage of disulfide linkages. In the case of peptides that contain labile peptide bonds, for example X-Pro sequences, preferential cleavage at these sites results in two linear chains linked by a disulfide bond, which then undergo further f...