Studying the S-nitrosylation of model peptides and eNOS protein by mass spectrometry

Taldone, Frank S.; Tummala, Monorama; Goldstein, Eric J.; Ryzhov, Victor; Ravi, Kandasamy; Black, Stephen M.

doi:10.1016/j.niox.2005.06.004

Cited by 36 publications

(28 citation statements)

References 27 publications

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“…The other cysteine involved in Zn binding, Cys 98, was detected as one of the nitrosylated sites by LC-MS and reported in our previous study (Taldone et al, 2005). We have not observed peptides containing this cysteine in MALDI-MS experiments.…”

Section: Determination Of S-nitrosylation Sites In Enos Treated With supporting

confidence: 75%

“…In our previous study we modified the nitrosylated cysteines with iodoacetamide, and hence it was easier to detect that eNOS peptide containing Cys 98 (Taldone et al, 2005). Nonetheless, the fact that both cysteines involved in the formation of eNOS dimer are found nitrosylated supports our previous finding that the monomerization of eNOS homodimer in the presence of NO donors might be due to the S-nitrosylation of Zn-binding cysteines (Ravi et al, 2004).…”

supporting

confidence: 57%

“…Though nitrosylated proteins and peptides are known to be highly labile during mass spectrometry analysis, modification of nitrosothiols with biotin gives the advantage of improved stability of nitrosylated peptides in a mass spectrometer and the ease of enriching these peptides with avidin columns (Kaneko and Yoshinao, 2003). The complete list of sites of S-nitrosylation identified in this study and previous study (Taldone et al, 2005) carried out by our group is shown in Table 1. It is likely that multiple factors account for the differences in modified cysteines detected in our experiments: variations in the extent of nitrosylation; differences in MS ionization techniques used along with the peptide ionization efficiency; as well as the potential for sample loss during the cleanup and elution from the neutravidin column.…”

Section: Determination Of S-nitrosylation Sites In Enos Treated With mentioning

confidence: 83%

See 2 more Smart Citations

Identification of the Cysteine Nitrosylation Sites in Human Endothelial Nitric Oxide Synthase

Tummala

Ryzhov

Ravi

et al. 2008

DNA and Cell Biology

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S-nitrosylation, or the replacement of the hydrogen atom in the thiol group of cysteine residues by a -NO moiety, is a physiologically important posttranslational modification. In our previous work we have shown that Snitrosylation is involved in the disruption of the endothelial nitric oxide synthase (eNOS) dimer and that this involves the disruption of the zinc (Zn) tetrathiolate cluster due to the S-nitrosylation of Cysteine 98. However, human eNOS contains 28 other cysteine residues whose potential to undergo S-nitrosylation has not been determined. Thus, the goal of this study was to identify the cysteine residues within eNOS that are susceptible to S-nitrosylation in vitro. To accomplish this, we utilized a modified biotin switch assay. Our modification included the tryptic digestion of the S-nitrosylated eNOS protein to allow the isolation of S-nitrosylated peptides for further identification by mass spectrometry. Our data indicate that multiple cysteine residues are capable of undergoing S-nitrosylation in the presence of an excess of a nitrosylating agent. All these cysteine residues identified were found to be located on the surface of the protein according to the available X-ray structure of the oxygenase domain of eNOS. Among those identified were Cys 93 and 98, the residues involved in the formation of the eNOS dimer through a Zn tetrathiolate cluster. In addition, cysteine residues within the reductase domain were identified as undergoing S-nitrosylation. We identified cysteines 660, 801, and 1113 as capable of undergoing S-nitrosylation. These cysteines are located within regions known to bind flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NADPH) although from our studies their functional significance is unclear. Finally we identified cysteines 852, 975=990, and 1047=1049 as being susceptible to Snitrosylation. These cysteines are located in regions of eNOS that have not been implicated in any known biochemical functions and the significance of their S-nitrosylation is not clear from this study. Thus, our data indicate that the eNOS protein can be S-nitrosylated at multiple sites other than within the Zn tetrathiolate cluster, suggesting that S-nitrosylation may regulate eNOS function in ways other than simply by inducing dimer collapse.

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Section: Determination Of S-nitrosylation Sites In Enos Treated With supporting

confidence: 75%

supporting

confidence: 57%

Section: Determination Of S-nitrosylation Sites In Enos Treated With mentioning

confidence: 83%

See 1 more Smart Citation

Identification of the Cysteine Nitrosylation Sites in Human Endothelial Nitric Oxide Synthase

Tummala

Ryzhov

Ravi

et al. 2008

DNA and Cell Biology

Self Cite

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“…However few recent studies have raised questions about presence of such consensus motif. Study by Taldone et al (2005) revealed that there is no profound effect of the primary sequence of surrounding amino acid residues on the reactivity of cysteines towards S-nitrosylation at peptide level. All the peptides independent of the amino acid surrounding the cysteine residue underwent rapid S-nitrosylation.…”

Section: S-nitrosylation Sensitive Motifsmentioning

confidence: 99%

S-Nitrosylation — another biological switch like phosphorylation?

Abat

Saigal

Deswal

2008

Physiol Mol Biol Plants

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Nitric oxide (NO) has emerged as a key-signaling molecule affecting plant growth and development right from seed germination to cell death. It is now being considered as a new plant hormone. NO is predominantly produced by nitric oxide synthase (NOS) in animal systems. NOS converts L-arginine (substrate) to citrulline and NO is a byproduct of the reaction. However, a similar biosynthetic mechanism is still not fully established in plants as NOS is still to be purified. First plant NOS gene (AtNOS1) was cloned from Arabidopsis suggesting the existence of NOS in plants. It was shown to be involved in hormonal signaling, stomatal closure, flowering, pathogen defense response, oxidative stress, senescence and salt tolerance. However, recent studies have raised critical questions/concerns about its substantial role in NO biosynthesis. Despite the ever increasing number of NO responses observed, little is known about the signal transduction pathway(s) and mechanisms by which NO interacts with different components and results in altered cellular activities. A brief overview is presented here. Proteins are one of the major bio-molecule besides DNA, RNA and lipids which are modified by NO and its derivatives. S-nitrosylation is a ubiquitous NO mediated posttranslational modification that might regulate broad spectrum of proteins. In this review S-nitrosylation formation, catabolism and its biological significance is discussed to present the current scenario of this modification in plants.

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“…Indeed, reports demonstrating the identification of peptide S-nitrosylation sites by LC/MS/MS have been published by Mirza et al, using a Finnegan-MAT TSQ-700 triple quadrupole instrument [25]; by Taldone et al, using a Bruker Daltonics Esquire 3000 quadrupole ion trap instrument [26]; by Chen et al using an ABI QSTAR-XL hybrid QTOF instrument [12]; and by Hao et al, using an Agilent Technologies MSD Ultra ion trap [27] and Micromass Quattro II triple quadrupole instruments [28]. Since each type of instrument is configured differently, MS parameters should be optimized for each S-nitrosylation study.…”

mentioning

confidence: 99%

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

Wang

Liu

et al. 2008

J. Am. Soc. Mass Spectrom.

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S-nitrosylation of proteins serves an important role in regulating diverse cellular processes including signal transduction, DNA repair, and neurotransmission. Identification of Snitrosylation sites is crucial for understanding the significance of this post-translational modification (PTM) in modulating the function of a protein. However, it is challenging to identify S-nitrosylation sites directly by mass spectrometric (MS) methods due to the labile nature of the 5-NO bond. Here we describe a strategy for direct identification of protein S-nitrosylation sites in an electrospray ionization (ESI) quadrupole time-of-flight (QTOF) mass spectrometer without prior chemical derivatization of S-nitrosylated peptides. Both sample buffer composition and MS hardware parameters were carefully adjusted to ensure that S-nitrosylated peptide ions could be analyzed by the QTOF MS with optimal signal/noise ratios. It was crucial that the proteins were preserved in a sample solution containing 1 mM EDTA and 0.1 mM neocuproine at neutral pH. Proteins dissolved in this solution are amenable to in-solution tryptic digestion, which is important for the analysis of biological samples. S-nitrosylated peptides were effectively analyzed by LC/MS/MS on QTOF MS, with an optimized cone voltage of 20 V and collision energy of 4 V. We have successfully applied this method to thioredoxin, a key antioxidant protein, and identified within it an S-nitrosylation site at

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Studying the S-nitrosylation of model peptides and eNOS protein by mass spectrometry

Cited by 36 publications

References 27 publications

Identification of the Cysteine Nitrosylation Sites in Human Endothelial Nitric Oxide Synthase

Identification of the Cysteine Nitrosylation Sites in Human Endothelial Nitric Oxide Synthase

S-Nitrosylation — another biological switch like phosphorylation?

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

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