2014年2月
Identification of nitrated tyrosine residues of protein kinase G-I alpha by mass spectrometry
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
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- 巻
- 406
- 号
- 5
- 開始ページ
- 1387
- 終了ページ
- 1396
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1007/s00216-013-7535-4
- 出版者・発行元
- SPRINGER HEIDELBERG
The nitration of tyrosine to 3-nitrotyrosine is an oxidative modification of tyrosine by nitric oxide and is associated with many diseases, and targeting of protein kinase G (PKG)-I represents a potential therapeutic strategy for pulmonary hypertension and chronic pain. The direct assignment of tyrosine residues of PKG-I has remained to be made due to the low sensitivity of the current proteomic approach. In order to assign modified tyrosine residues of PKG-I, we nitrated purified PKG-I alpha expressed in insect Sf9 cells by use of peroxynitrite in vitro and analyzed the trypsin-digested fragments by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Among the 21 tyrosine residues of PKG-I alpha, 16 tyrosine residues were assigned in 13 fragments; and six tyrosine residues were nitrated, those at Y71, Y141, Y212, Y336, Y345, and Y567, in the peroxynitrite-treated sample. Single mutation of tyrosine residues at Y71, Y212, and Y336 to phenylalanine significantly reduced the nitration of PKG-I alpha; and four mutations at Y71, Y141, Y212, and Y336 (Y4F mutant) reduced it additively. PKG-I alpha activity was inhibited by peroxynitrite in a concentration-dependent manner from 30 mu M to 1 mM, and this inhibition was attenuated in the Y4F mutant. These results demonstrated that PKG-I alpha was nitrated at multiple tyrosine residues and that its activity was reduced by nitration of these residues.
- リンク情報
- ID情報
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- DOI : 10.1007/s00216-013-7535-4
- ISSN : 1618-2642
- eISSN : 1618-2650
- Web of Science ID : WOS:000331004700012