Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1217, Issue 25, Pages 4120-4126Publisher
ELSEVIER
DOI: 10.1016/j.chroma.2010.02.013
Keywords
Phytochelatins; S-Nitrosylation; Direct electrospray-mass spectrometry analysis
Funding
- MIUR (Ministero dell'Istruzione, dell'Universita e della Ricerca, Italy)
- Sviluppo di metodologie innovative per l'analisi di prodotti agroalimentari [RBIP06SXMR]
- [PNR 2007-2010]
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Reversed-phase liquid chromatography (RPLC) and electrospray (ESI)-linear ion trap (LIT) mass spectrometry was applied to the direct characterization of in vivo S-nitrosylated (SNO) phytochelatins (PCs) expressed in cadmium-stressed Arabidopsis thaliana cells. Cys-nitrosylation is under discussion as in vivo redox-based post-translational modification of proteins and peptides in plants in which the -NO group is involved as signal molecule in different biological functions. The gas-phase ion chemistry of in vivo and in vitro generated SNO-PCs was compared with the aim of evaluating NO binding stability and improving MS knowledge about peptide nitrosation. Using RPLC separation and ESI-LIT-MS, mono-nitrosylated PCs were identified in in vivo cadmium treated A. thaliana cells without derivatization. The in vivo binding of the NO group to PC2, PC3 and PC4 resulted to occur selectively on only one cystein residue. The fragmentation pathway energies of the in vitro GSNO-generated NO-PCs with respect to the in vivo NO-PCs were investigated, suggesting the presence of a different internal stability for these molecules. By carrying out MS2 experiments on these quasi-symmetric peptides, the different stability degree of the NO group was demonstrated to be correlated with the PC chain length. In addition, the data obtained highlight a putative role of the adjacent Glu/Cys motif in the gas-phase stability of the NO-containing molecule. (C) 2010 Elsevier B.V. All rights reserved.
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