Disulfide structures of highly bridged peptides: A new strategy for analysis

Gray, William R.

doi:10.1002/pro.5560021017

Cited by 240 publications

(273 citation statements)

References 34 publications

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“…Although every disulfide bond has the same redlox POtential and should be reduced to a similar extent from the standpoint of thermodynamics, the reduction kinetics of various disulfide bonds in solutions of native or partially denatured protein are quite different, depending on accessibility of the disulfide bonds to reducing agents and on the conformational energy of the disulfide bonds (Reeve & Pierce, 1981;Kuwajimaet al, 1990;Gray, 1993a;Takeda et al, 1995). Our methodology requires that individual disulfide bonds be broken at comparable rates.…”

Section: Partial Reduction Of Proteinsmentioning

confidence: 99%

“…Water-soluble TCEP has proved to be an excellent reducing agent for disulfide bonds (Gray, 1993a(Gray, , 1993bWu et al, 1996). Reduction by TCEP can be carried out at pH 3.0 to suppress disulfide bond scrambling.…”

Section: Partial Reduction Of Proteinsmentioning

confidence: 99%

“…Reduction by TCEP can be carried out at pH 3.0 to suppress disulfide bond scrambling. Furthermore, at pH 3.0, the reduction of disulfide bonds is kinetically controlled, which makes partial reduction possible (Gray, 1993a).…”

Section: Partial Reduction Of Proteinsmentioning

confidence: 99%

“…Although supplementary methodologies such as non-specific fragmentation by partial acid hydrolysis have been proposed to avoid disulfide bond scrambling, it is difficult to deal with the data obtained by these nonspecific techniques. Gray (1993aGray ( , 1993b has described an approach for analyzing disulfide linkage patterns in highly bridged small peptides with close or adjacent cysteine residues. In his experiments, peptides were partially reduced, the nascent free thiols alkylated, and their positions recognized from the results of sequence analysis.…”

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confidence: 99%

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A novel methodology for assignment of disulfide bond pairings in proteins

1997

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A novel methodology is described for the assignment of disulfide bonds in proteins of known sequence. The denatured protein is subjected to limited reduction by tris(2-carboxyethy1)phosphine (TCEP) in pH 3.0 citrate buffer to produce a mixture of partially reduced protein isomers; the nascent sulfhydryls are immediately cyanylated by I-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP) under the same buffered conditions. The cyanylated protein isomers, separated by and collected from reversed-phase HPLC, are subjected to cleavage of the peptide bonds on the N-terminal side of cyanylated cysteines in aqueous ammonia to form truncated peptides that are still linked by residual disulfide bonds. The remaining disulfide bonds are then completely reduced to give a mixture of peptides that can be mass mapped by MALDI-MS. The masses of the resulting peptide fragments are related to the location of the paired cysteines that had undergone reduction, cyanylation, and cleavage. A side reaction, p-elimination, often accompanies cleavage and produces overlapped peptides that provide complementary confirmation for the assignment. This strategy minimizes disulfide bond scrambling and is simple, fast, and sensitive. The feasibility of the new approach is demonstrated in the analysis of model proteins that contain various disulfide bond linkages, including adjacent cysteines. Experimental conditions are optimized for protein partial reduction, sulfhydryl cyanylation, and chemical cleavage reactions. Keywords: chemical cleavage; cyanylation; disulfide bonds; MALDI-MS; partial reductionAlthough recombinant DNA techniques have made available an increasing number of deduced protein sequences, they do not provide structural information regarding post-translational modifications. Disulfide bonding in proteins is one of the frequently encountered post-translational modifications. Disulfide bonds can play an important role in establishing and maintaining some three-dimensional structures. The assignment of disulfide bonds is, therefore, an important aspect in the structural characterization of proteins. However, although there are good methods for quantifying the number of disulfide bonds in proteins, the unambiguous determination of the location or pairing of disulfide bonds continues to challenge protein chemists.

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Section: Partial Reduction Of Proteinsmentioning

confidence: 99%

Section: Partial Reduction Of Proteinsmentioning

confidence: 99%

Section: Partial Reduction Of Proteinsmentioning

confidence: 99%

mentioning

confidence: 99%

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A novel methodology for assignment of disulfide bond pairings in proteins

1997

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“…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.…”

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confidence: 99%

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

Thakur

Balaram

2009

J. Am. Soc. Mass Spectrom.

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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 ...

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Distinction between the three disulfide isomers of guanylin 99-115 by low-energy collision-induced dissociation

Badock

Raida²,

Adermann³

et al. 1998

Rapid Commun. Mass Spectrom.

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Guanylin 99-115 is a small human peptide hormone with two disulfide bonds. The four cysteinyl residues in this peptide allow the formation of two disulfide bridges in three different ways but only the 1-3/2-4 combination is able to bind to the receptor and cause an increase of intracellular cGMP, while the other isomers are biologically inactive. Using guanylin 99-115 as a model peptide, the aim of this study was to investigate whether it is possible to distinguish the differently bridged isomers directly by tandem mass spectrometry. Guanylin isomers were generated by performing an air oxidation of fully reduced guanylin 1-3/2-4 obtained by chemical synthesis. The reaction product is a mixture of guanylin 1-4/2-3 and guanylin 1-2/3-4 in a ratio of 3:1, but there is virtually no guanylin 1-3/2-4. The two biologically inactive peptides were separated by reversed-phase high pressure liquid chromatography (HPLC). Using low-energy collision-induced dissociation tandem mass spectrometry, it was possible to distinguish unambiguously between the three guanylin isomers. This was possible due to the identification of a large number of fragments with intact disulfide bonds. Accordingly, this strategy of a direct and sensitive analysis should work as well for other peptides, with the potential to determine an undefined disulfide bond pattern.

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Disulfide structures of highly bridged peptides: A new strategy for analysis

Cited by 240 publications

References 34 publications

A novel methodology for assignment of disulfide bond pairings in proteins

A novel methodology for assignment of disulfide bond pairings in proteins

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

Distinction between the three disulfide isomers of guanylin 99-115 by low-energy collision-induced dissociation

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