A strategy for direct identification of protein S-nitrosylation sites by quadrupole time-of-flight mass spectrometry

Wang, Yan; Liu, Tong; Wu, Changgong; Li, Hong

doi:10.1016/j.jasms.2008.06.001

Cited by 73 publications

(59 citation statements)

References 34 publications

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“…Losses of HNO and HONO ϩ CO, which are indicative of N-nitrosoamino acids [41], are not observed. These results are in full agreement with previous studies from our [40] and other [28,42] groups, which highlighted the labile character of S-NO bond under various MS conditions. In fact, most studies involving S-nitrosylated peptides focus on optimizing the MS conditions to prevent the loss of NO from nitrosylated Cys residues [40,42].…”

Section: Fragmentation Of Protonated S-nitrosocysteinesupporting

confidence: 93%

“…These results are in full agreement with previous studies from our [40] and other [28,42] groups, which highlighted the labile character of S-NO bond under various MS conditions. In fact, most studies involving S-nitrosylated peptides focus on optimizing the MS conditions to prevent the loss of NO from nitrosylated Cys residues [40,42]. Numerous approaches have been developed for covalent modification of the nitrosothiol moiety (including the biotin switch assay [43,44] to avoid the fragmentation of the S-NO bond during MS-based analysis [45,46].…”

Section: Fragmentation Of Protonated S-nitrosocysteinesupporting

confidence: 93%

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Gas-phase fragmentation of long-lived cysteine radical cations formed via no loss from protonated S-nitrosocysteine

Ryzhov

Lam

O’Hair

2009

J. Am. Soc. Mass Spectrom.

101

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In this work, we describe two different methods for generating protonated S-nitrosocysteine in the gas phase. The first method involves a gas-phase reaction of protonated cysteine with t-butylnitrite, while the second method uses a solution-based transnitrosylation reaction of cysteine with S-nitrosoglutathione followed by transfer of the resulting S-nitrosocysteine into the gas phase by electrospray ionization mass spectrometry (ESI-MS). Independent of the way it was formed, protonated S-nitrosocysteine readily fragments via bond homolysis to form a long-lived radical cation of cysteine (Cys •ϩ ), which fragments under collision-induced dissociation (CID) conditions via losses in the following relative abundance order: •COOH ӷ CH 2 S Ͼ •CH 2 SH Ϸ H 2 S. Deuterium labeling experiments were performed to study the mechanisms leading to these pathways. DFT calculations were also used to probe aspects of the fragmentation of protonated S-nitrosocysteine and the radical cation of cysteine. NO loss is found to be the lowest energy channel for the former ion, while the initially formed distonic Cys•ϩ with a sulfur radical site undergoes proton and/or H atom transfer reactions that precede the losses of T here has been renewed interest in the gas-phase formation and reactions of radical ions of biomolecules. The motivation for these studies range from fundamental interest in species related to biological processes such as enzyme catalysis [1] and oxidative chemistry associated with damage to biomolecules such as DNA [2] and proteins [3] through to the potential for developing novel mass spectrometry based analytical applications [4]. With regards to the formation of radical ions of amino acids and peptides, several methods have been developed as alternatives to electron ionization (EI) [5]. These include UV photodissociation [6 -11] and ion-electron based techniques such as electron capture dissociation (ECD) [12][13][14][15]. Chemical-based methods that involve low-energy collisioninduced dissociation (CID) [16] of ions generated via electrospray ionization and which can thus be carried out on a wide range of mass spectrometers, merit special discussion. The first, pioneered by Siu, involves carrying out CID of ternary metal complexes to form radical cations of peptides via the redox reaction shown in eq 1 [17][18][19]. To date, doubly charged copper(II) ternary metal complexes (eq 1, where Metal ϭ Cu; L ϭ a range of neutral ligands, x ϭ 2, y ϭ 0) and singly charged metal(III) complexes (eq 1, where Metal ϭ Cr, Mn, Fe, and Co; L ϭ a salen ligand, x ϭ 3, y ϭ 2) have been used to study the formation and reactions of peptide radical ions, including recent contributions from Julia Laskin [20 -23].The second method involves carrying out CID on a cationized peptide containing a functional group with a weak bond, which is susceptible to bond homolysis. A number of different functional groups have been examined to date including: (1) N-terminal azo derivatives

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Section: Fragmentation Of Protonated S-nitrosocysteinesupporting

confidence: 93%

Section: Fragmentation Of Protonated S-nitrosocysteinesupporting

confidence: 93%

Gas-phase fragmentation of long-lived cysteine radical cations formed via no loss from protonated S-nitrosocysteine

Ryzhov

Lam

O’Hair

2009

J. Am. Soc. Mass Spectrom.

101

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“…S-nitrosylation procedure was adapted from [5,7]: 0.3 mg of each peptide was dissolved in 900 μL of 1 mM EDTA (Fisher Scientific, Leicestershire, UK) and 0.1 mM neocuproine (Sigma-Aldrich, Dorset, UK), and reacted with 100 μL 1 mM GSNO (Sigma-Aldrich) for 30 min at 39°C in the dark. The reaction was quenched by freezing at −80°C and no further purification was completed.…”

Section: Preparation Of Synthetic Peptidesmentioning

confidence: 99%

“…The characterization of S-nitrosylation is technically challenging, and methods have been reviewed by Torta et al [6]. Li and co-workers described an electrospray Q-TOF strategy for direct analysis of S-nitrosylation, which relied on specific buffer conditions and mass spectrometry parameters [7]. Alternatively, the S-NO can be converted to an S-detectable tag e.g., the biotin switch method [8], the SNOSID (SNO Site IDentification) method [9], or the SHIPS (Site-specific High-throughput Identification of Protein S-nitrosylation) method [10].…”

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confidence: 99%

The Radical Ion Chemistry of S-Nitrosylated Peptides

Jones

Winn

Cooper

2012

J. Am. Soc. Mass Spectrom.

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The radical ion chemistry of a suite of S-nitrosopeptides has been investigated. Doubly and triply-protonated ions of peptides NYCGLPGEYWLGNDK, NYCGLPGEYWLGNDR, NYCGLP-GERWLGNDR, NACGAPGEKWAGNDK, NYCGLPGEKYLGNDK, NYGLPGCEKWYGNDK and NYGLPGEKWYGCNDK were subjected to electron capture dissociation (ECD), and collisioninduced dissociation (CID). The peptide sequences were selected such that the effect of the site of S-nitrosylation, the nature and position of the basic amino acid residues, and the nature of the other amino acid side chains, could be interrogated. The ECD mass spectra were dominated by a peak corresponding to loss of• NO from the charge-reduced precursor, which can be explained by a modified Utah-Washington mechanism. Some backbone fragmentation in which the nitrosyl modification was preserved was also observed in the ECD of some peptides. Molecular dynamics simulations of peptide ion structure suggest that the ECD behavior was dependent on the surface accessibility of the protonated residue. CID of the S-nitrosylated peptides resulted in homolysis of the S-N bond to form a long-lived radical with loss of • NO. The radical peptide ions were isolated and subjected to ECD and CID. ECD of the radical peptide ions provided an interesting comparison to ECD of the unmodified peptides. The dominant process was electron capture without further dissociation (ECnoD). CID of the radical peptide ions resulted in cysteine, leucine, and asparagine side chain losses, and radical-induced backbone fragmentation at tryptophan, tyrosine, and asparagine residues, in addition to charge-directed backbone fragmentation.

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“…Each of these has been implicated in S-nitrosation activities (also called S-nitrosylation), particularly with respect to regulation of apoptosis. [7][8][9][10][11][12][13][14] S-nitrosation/nitrosylation is the addition of NO to a cysteine residue, a redox reaction requiring one electron oxidation, and numerous proteins have been suggested to be regulated in this manner. 15 Removal of the nitroso group in target proteins, or transfer of the group to other proteins, is likely to involve hTrx1 in human cells.…”

Section: Introductionmentioning

confidence: 99%

Crystal structure of human thioredoxin revealing an unraveled helix and exposed S‐nitrosation site

Weichsel¹,

Kem²,

Montfort³

2010

Protein Science

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Thioredoxins reduce disulfide bonds and other thiol modifications in all cells using a CXXC motif. Human thioredoxin 1 is unusual in that it codes for an additional three cysteines in its 105 amino acid sequence, each of which have been implicated in other reductive activities. Cys 62 and Cys 69 are buried in the protein interior and lie at either end of a short helix (helix 3), and yet can disulfide link under oxidizing conditions. Cys 62 is readily S-nitrosated, giving rise to a SNO modification, which is also buried. Here, we present two crystal structures of the C69S/C73S mutant protein under oxidizing (1.5 Å ) and reducing (1.1 Å ) conditions. In the oxidized structure, helix 3 is unraveled and displays a new conformation that is stabilized by a series of new hydrogen bonds and a disulfide link with Cys 62 in a neighboring molecule. The new conformation provides an explanation for how a completely buried residue can participate in SNO exchange reactions.

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A strategy for direct identification of protein S-nitrosylation sites by quadrupole time-of-flight mass spectrometry

Cited by 73 publications

References 34 publications

Gas-phase fragmentation of long-lived cysteine radical cations formed via no loss from protonated S-nitrosocysteine

Gas-phase fragmentation of long-lived cysteine radical cations formed via no loss from protonated S-nitrosocysteine

The Radical Ion Chemistry of S-Nitrosylated Peptides

Crystal structure of human thioredoxin revealing an unraveled helix and exposed S‐nitrosation site

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