Reversible Hydrogen Transfer between Cysteine Thiyl Radical and Glycine and Alanine in Model Peptides: Covalent H/D Exchange, Radical−Radical Reactions, and <scp>l</scp>- to <scp>d</scp>-Ala Conversion

Mozziconacci, Olivier; Kerwin, Bruce A.; Schöneich, Christian

doi:10.1021/jp101508b

Cited by 33 publications

(50 citation statements)

References 52 publications

(92 reference statements)

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“…Strategy. Our strategy to identify D-amino acid containing sequences in photoirradiated mAb1 followed the methodology that we had applied to characterize C−H bonds sensitive to light-induced covalent H/D exchange, 3,[27][28][29]34,35 and which Huang et al 32 and Amano et al 33 had applied to detect pH-dependent epimerization in several IgG variants. In initial experiments, mAb1 was dissolved in D 2 O and lightinduced incorporation of deuterium into the peptide map of mAb1 monitored by HPLC−MS and HPLC−MS/MS analysis.…”

Section: Resultsmentioning

confidence: 99%

Sequence-Specific Formation of d-Amino Acids in a Monoclonal Antibody during Light Exposure

Mozziconacci

Schöneich

2014

Mol. Pharmaceutics

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The photoirradiation of a monoclonal antibody 1 (mAb1) at λ = 254 nm and λmax = 305 nm resulted in the sequence-specific generation of d-Val, d-Tyr, and potentially d-Ala and d-Arg, in the heavy chain sequence [95-101] YCARVVY. d-Amino acid formation is most likely the product of reversible intermediary carbon-centered radical formation at the (α)C-positions of the respective amino acids ((α)C(•) radicals) through the action of Cys thiyl radicals (CysS(•)). The latter can be generated photochemically either through direct homolysis of cystine or through photoinduced electron transfer from Trp and/or Tyr residues. The potential of mAb1 sequences to undergo epimerization was first evaluated through covalent H/D exchange during photoirradiation in D2O, and proteolytic peptides exhibiting deuterium incorporation were monitored by HPLC-MS/MS analysis. Subsequently, mAb1 was photoirradiated in H2O, and peptides, for which deuterium incorporation in D2O had been documented, were purified by HPLC and subjected to hydrolysis and amino acid analysis. Importantly, not all peptide sequences which incorporated deuterium during photoirradiation in D2O also exhibited photoinduced d-amino acid formation. For example, the heavy chain sequence [12-18] VQPGGSL showed significant deuterium incorporation during photoirradiation in D2O, but no photoinduced formation of d-amino acids was detected. Instead this sequence contained ca. 22% d-Val in both a photoirradiated and a control sample. This observation could indicate that d-Val may have been generated either during production and/or storage or during sample preparation. While sample preparation did not lead to the formation of d-Val or other d-amino acids in the control sample for the heavy chain sequence [95-101] YCARVVY, we may have to consider that during hydrolysis N-terminal residues (such as in VQPGGSL) may be more prone to epimerization. We conclude that the photoinduced, radical-dependent formation of d-amino acids requires not only the intermediary formation of a (α)C(•) radical but also sufficient flexibility of the protein domain to allow both pro-chiral faces of the (α)C(•) radical to accept a hydrogen atom.

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Section: Resultsmentioning

confidence: 99%

Sequence-Specific Formation of d-Amino Acids in a Monoclonal Antibody during Light Exposure

Mozziconacci

Schöneich

2014

Mol. Pharmaceutics

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“…This observation indicates hydrogen transfer from the first two amino acid residues to the thiyl radical. In fact, experimental and theoretical studies have shown that intramolecular hydrogen transfer to the thiyl radical is a facile process within polypeptides and cysteine ions [30][31][32][33]. [34] or CID of Nterminus amidated peptides [35].…”

Section: Formation Of C Z Ionsmentioning

confidence: 99%

Electron Transfer Dissociation (ETD) of Peptides Containing Intrachain Disulfide Bonds

Cole

Zhang

et al. 2011

J. Am. Soc. Mass Spectrom.

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The fragmentation chemistry of peptides containing intrachain disulfide bonds was investigated under electron transfer dissociation (ETD) conditions. Fragments within the cyclic region of the peptide backbone due to intrachain disulfide bond formation were observed, including: c (odd electron), z (even electron), c-33 Da, z+33 Da, c+32 Da, and z-32 Da types of ions. The presence of these ions indicated cleavages both at the disulfide bond and the N-Cα backbone from a single electron transfer event. Mechanistic studies supported a mechanism whereby the N-Cα bond was cleaved first, and radical-driven reactions caused cleavage at either an S-S bond or an S-C bond within cysteinyl residues. Direct ETD at the disulfide linkage was also observed, correlating with signature loss of 33 Da (SH) from the charge-reduced peptide ions. Initial ETD cleavage at the disulfide bond was found to be promoted amongst peptides ions of lower charge states, while backbone fragmentation was more abundant for higher charge states. The capability of inducing both backbone and disulfide bond cleavages from ETD could be particularly useful for sequencing peptides containing intact intrachain disulfide bonds. ETD of the 13 peptides studied herein all showed substantial sequence coverage, accounting for 75%-100% of possible backbone fragmentation.

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“…However, these HAT reactions occurred not only with the α C-H bonds but also with the C-H bonds of the amino acid side chains[29]. Radiation chemistry, ESR, NMR and mass spectrometry experiments then provided evidence for intra molecular HAT reactions in model peptides[30–32] as well as in glutathione (GSH)[33–38], where HAT was observed between the Cys thiyl radical and the α C-H bonds of γ-Glu, Cys and Gly. More recently, pulse radiolysis experiments with Cys and several model compounds provided rate constants for intra molecular HAT reactions with the α C-H and the β C-H bond (Scheme 1, equilibria 2 and 1)[39].…”

Section: Introductionmentioning

confidence: 99%

“…While thiyl radicals were not generated through physiological processes in these studies, they are relevant to physiology as they demonstrate what can happen when thiyl radicals are generated on proteins. In the absence of oxygen, the reversible generation of carbon-centered radicals has led to the conversion of L-Ala to D-Ala in a model peptide[32], and to the sequence-specific generation of D-amino acids on IgG1[51]. Hence, thiyl radical generation on proteins is a viable source for D-amino acid generation, and such reactions have to be taken into account when simulating thiyl radical reaction pathways in proteins (see below).…”

Section: Introductionmentioning

confidence: 99%

Protein thiyl radical reactions and product formation: a kinetic simulation

Nauser

Koppenol

Schöneich

2015

Free Radical Biology and Medicine

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Protein thiyl radicals are important intermediates generated in redox processes of thiols and disulfides. Thiyl radicals efficiently react with glutathione and ascorbate, and the common notion is that these reactions serve to eliminate thiyl radicals before they can enter potentially hazardous processes. However, over the past years increasing evidence has been provided for rather efficient intramolecular hydrogen transfer processes of thiyl radicals in proteins and peptides. Based on rate constants published for these processes, we have performed kinetic simulations of protein thiyl radical reactivity. Our simulations suggest that protein thiyl radicals enter intramolecular hydrogen transfer reactions to a significant extent even under physiologic conditions, i.e. in the presence of 30 μM oxygen, 1 mM ascorbate and 10 mM glutathione. At lower concentrations of ascorbate and glutathione, frequently observed when tissue is exposed to oxidative stress, the extent of irreversible protein thiyl radical-dependent protein modification increases.

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Reversible Hydrogen Transfer between Cysteine Thiyl Radical and Glycine and Alanine in Model Peptides: Covalent H/D Exchange, Radical−Radical Reactions, and l- to d-Ala Conversion

Cited by 33 publications

References 52 publications

Sequence-Specific Formation of d-Amino Acids in a Monoclonal Antibody during Light Exposure

Sequence-Specific Formation of d-Amino Acids in a Monoclonal Antibody during Light Exposure

Electron Transfer Dissociation (ETD) of Peptides Containing Intrachain Disulfide Bonds

Protein thiyl radical reactions and product formation: a kinetic simulation

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