A strategy for direct identification of protein S-nitrosylation sites by quadrupole time-of-flight mass spectrometry.
S-nitrosylation of proteins serves an important role in regulating diverse cellular processes including signal transduction, 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.
Wang
Yan
Y
Center for Advanced Proteomics Research and Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School Cancer Center, Newark, New Jersey, USA.
Liu
Tong
T
Wu
Changgong
C
Li
Hong
H
D016229:Amyloid beta-Peptides / Q000737:chemistry; D001665:Binding Sites; D002614:Chelating Agents / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D006801:Humans; D007328:Insulin / Q000737:chemistry; D008832:Microchemistry; D009603:Nitroso Compounds / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D010618:Phenanthrolines / Q000737:chemistry; D026422:S-Nitrosoglutathione / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry
D016229:Amyloid beta-Peptides; D002614:Chelating Agents; D007328:Insulin; D009603:Nitroso Compounds; D010446:Peptide Fragments; D010455:Peptides; D010618:Phenanthrolines; C097877:insulin B (9-23); D026422:S-Nitrosoglutathione; C002701:neocuproine; D004492:Edetic Acid