Confirmation by Mass Spectrometry of a Trisulfide Variant in Methionyl Human Growth Hormone Biosynthesized in<i>Escherichia coli</i>

Canova-Davis, Eleanor; Baldonado, Ida P.; Chloupek, Rosanne C.; Ling, Victor; Gehant, Richard L.; Olson, Kenneth; Gillece-Castro, Beth

doi:10.1021/ac9605915

Cited by 38 publications

(27 citation statements)

References 45 publications

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“…[7,8] More recently, Tous et al reported the occurrence of a nonreducible thioether bridge between the light and heavy chains of different IgG1 monoclonal antibodies. [9] However, the present article is to our knowledge the first description of a thioether-containing hGH variant.…”

Section: Discussionmentioning

confidence: 99%

Characterisation of a Novel Growth Hormone Variant Comprising a Thioether Link between Cys182 and Cys189

et al. 2007

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A novel variant of recombinant human growth hormone (r-hGH), isolated from biopharmaceutical preparations produced in E. coli, was identified and characterised. This variant contains a nonreducible thioether bridge near the C terminus between Cys182 and Cys189 and was characterised using various analytical techniques. As previous work by Cunningham and Wells (1993) highlighted the involvement of several residues in this part of the sequence in the binding and affinity of the molecule to its receptor, the presence of this modified intramolecular link may have important implications with regard to the biological behaviour of the molecule. Furthermore, as the conversion of a disulfide into a thioether was previously reported for a therapeutic monoclonal antibody (Tous et al., 2005), this may imply that disulfide bridges located in this part of the molecule have a generic susceptibility to thioether formation. This in turn is relevant to the biopharmaceutical industry for monitoring the integrity of disulfide bridges near the protein C terminus. The present study exhibits a state of the art physicochemical investigation for the unequivocal elucidation of a novel structure involving peptide mapping with mass spectrometry and de novo peptide sequencing. Changes in the higher order structure of the molecule were highlighted by near UV circular dichroism and molecular modelling.

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Section: Discussionmentioning

confidence: 99%

Characterisation of a Novel Growth Hormone Variant Comprising a Thioether Link between Cys182 and Cys189

et al. 2007

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“…For proteins, the modification was first discovered in cysteine bridges (Cys 53 -Cys 165 and Cys 182 -Cys 189 ) of biosynthetic human growth hormone produced in Escherichia coli [1,2] and in the non-recombinant protein Cu-Zn superoxide dismutase [3]. More recently, trisulfides have been identified and characterized in recombinant monoclonal antibodies (mAbs) of all immunoglobulin G (IgG) sub-classes [4][5][6][7].…”

Section: Introductionmentioning

confidence: 99%

A high-throughput hydrophilic interaction liquid chromatography coupled with a charged aerosol detector method to assess trisulfides in IgG1 monoclonal antibodies using tris(2-carboxyethyl)phosphine reaction products: Tris(2-carboxyethyl)phosphine-oxide and tris(2-carboxyethyl)phosphine-sulfide

Cornell¹,

Karanjit²,

Chen³

et al. 2016

Journal of Chromatography A

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“…Interestingly, reports of trisulfide formation when proteins are exposed to alkaline treatments have also appeared [54]. The occurrence of a trisulfide moiety in a recombinant human growth hormone has been established by mass spectrometry [55,56]. The high reactivity of the species formed when disulfides are exposed to basic media prompted us to re-examine the various products generated upon disulfide cleavage, by mass spectrometry.…”

mentioning

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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Confirmation by Mass Spectrometry of a Trisulfide Variant in Methionyl Human Growth Hormone Biosynthesized inEscherichia coli

Cited by 38 publications

References 45 publications

Characterisation of a Novel Growth Hormone Variant Comprising a Thioether Link between Cys182 and Cys189

Characterisation of a Novel Growth Hormone Variant Comprising a Thioether Link between Cys182 and Cys189

A high-throughput hydrophilic interaction liquid chromatography coupled with a charged aerosol detector method to assess trisulfides in IgG1 monoclonal antibodies using tris(2-carboxyethyl)phosphine reaction products: Tris(2-carboxyethyl)phosphine-oxide and tris(2-carboxyethyl)phosphine-sulfide

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

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