Mass spectrometry
Two synthesized peptides, MR21 (MCGGAIISDFIPAAAAVGGSR) and CR20
(CGGAIISDFIPAAAAVGGSR) (Proteogenix, Schiltigheim, France) were treated
with 0, 5, 10, 50, 100 and 500 μM of Diethylamine NONOate
(Sigma-Aldrich, USA) for 5 min at RT. The sample is taken up in
acetonitrile and formic acid (50% and 0.1% final respectively) and
immediately injected into the ionization source of an ESI-Q-TOF mass
spectrometer (micrOTOF-Q II, Brucker Daltonics, Germany) precalibrated
with a mixture of standard peptides (ESI-Low concentration Tunning Mix,
Agilent Technologies, USA). Data were acquired in positive ionisation
and in MS and MS/MS scan modes. Analysis parameters were optimized to
maintain and to be able to detect a maximum of Cys modifications,
particularly S-nitrosylation. The source temperature was 180°C, the
spray voltage was 5 kV, nebuliser gas (nitrogen) was set at 2.9 bar, and
dry gaz (nitrogen) was set at 10 L/min. Mass spectra acquisition
corresponded to 5000 spectra/sec on a mass range of 50e3000 m/z. LC-MS
raw data were processed using Data Analysis software (ESI Compass 1.3,
Bruker Daltonics, Germany). The identification of peptides was performed
using Biotools 3.2 and Sequence Editor Bruker’s software. Collected
spectrum data were compiled and presented with the SigmaPlot 10.0
software (Systat Software Inc, USA).