期刊
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
卷 19, 期 9, 页码 1353-1360出版社
AMER CHEMICAL SOC
DOI: 10.1016/j.jasms.2008.06.001
关键词
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资金
- NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE [P30NS046593] Funding Source: NIH RePORTER
- NINDS NIH HHS [P30 NS046593, NS046593, P30 NS046593-04] Funding Source: Medline
S-nitrosylation of proteins serves an important role in regulating diverse cellular processes including signal tranduction, DNA repair, and neurotransmission. Identification of S-nitrosylation 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 S-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 Cys73. (J Am Soc Mass Spectrom 2008, 19, 1353-1360) (C) 2008 American Society for Mass Spectrometry.
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