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

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

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Oxidative addition of a nitric oxide (NO) molecule to the thiol group of cysteine residues is a physiologically important post-translational modification that has been implicated in several metabolic and pathophysiological events. Our previous studies have indicated that S-nitrosylation can result in the disruption of the endothelial NO synthase (eNOS) dimer. It has been suggested that for S-nitrosylation to occur, the cysteine residue must be flanked by hydrophilic residues either in the primary structure or in the spatial proximity through appropriate conformation. However, this hypothesis has not been confirmed. Thus, the objective of this study was to determine if the nature of the amino acid residues that flank the cysteine in the primary structure has a significant effect on the rate and/or specificity of S-nitrosylation. To accomplish this, we utilized several model peptides based on the eNOS protein sequence. Some of these peptides contained point mutations to allow for different combinations of amino acid properties (acidic, basic, and hydrophobic) around the cysteine residue. To ensure that the results obtained were not dependent on the nitrosylation procedure, several common S-nitrosylation techniques were used and S-nitrosylation followed by mass spectrometric detection. Our data indicated that all peptides independent of the amino acids surrounding the cysteine residue underwent rapid S-nitrosylation. Thus, there does not appear to be a profound effect of the primary sequence of adjacent amino acid residues on the rate of cysteine S-nitrosylation at least at the peptide levels. Finally, our studies using recombinant human eNOS confirm that Cys98 undergoes S-nitrosylation. Thus, our data validate the importance of Cys98 in regulating eNOS dimerization and activity, and the utility of mass spectroscopy to identify cysteine residues susceptible to S-nitrosoylation.

Original languageEnglish (US)
Pages (from-to)176-187
Number of pages12
JournalNitric Oxide - Biology and Chemistry
Volume13
Issue number3
DOIs
StatePublished - Nov 1 2005

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Nitric Oxide Synthase
Mass spectrometry
Cysteine
Mass Spectrometry
Peptides
Proteins
Amino Acids
Acidic Amino Acids
Dimerization
Nitric Oxide Synthase Type III
Post Translational Protein Processing
Point Mutation
Sulfhydryl Compounds
Dimers
Conformations
Amino Acid Sequence
Nitric Oxide
Spectroscopy
Molecules

Keywords

  • Amino acid substitution
  • Cysteine modification
  • Endothelium
  • Mass spectrometric detection
  • Model peptide
  • Nitric oxide
  • Nitric oxide synthase
  • Recombinant protein
  • S-Nitrosylation
  • Zinc thiolate

ASJC Scopus subject areas

  • Biochemistry
  • Physiology
  • Clinical Biochemistry
  • Cancer Research

Cite this

Taldone, F. S., Tummala, M., Goldstein, E. J., Ryzhov, V., Ravi, K., & Black, S. M. (2005). Studying the S-nitrosylation of model peptides and eNOS protein by mass spectrometry. Nitric Oxide - Biology and Chemistry, 13(3), 176-187. https://doi.org/10.1016/j.niox.2005.06.004

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 Matthew.

In: Nitric Oxide - Biology and Chemistry, Vol. 13, No. 3, 01.11.2005, p. 176-187.

Research output: Contribution to journalArticle

Taldone, FS, Tummala, M, Goldstein, EJ, Ryzhov, V, Ravi, K & Black, SM 2005, 'Studying the S-nitrosylation of model peptides and eNOS protein by mass spectrometry', Nitric Oxide - Biology and Chemistry, vol. 13, no. 3, pp. 176-187. https://doi.org/10.1016/j.niox.2005.06.004
Taldone, Frank S. ; Tummala, Monorama ; Goldstein, Eric J. ; Ryzhov, Victor ; Ravi, Kandasamy ; Black, Stephen Matthew. / Studying the S-nitrosylation of model peptides and eNOS protein by mass spectrometry. In: Nitric Oxide - Biology and Chemistry. 2005 ; Vol. 13, No. 3. pp. 176-187.
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