Biology Reference
In-Depth Information
Chapter 41
Simultaneous Identifi cation and Quantifi cation
of Nitrosylation Sites by Combination of Biotin Switch
and ICAT Labeling
Abasse Fares , Claude Nespoulous , Michel Rossignol ,
and Jean-Benoît Peltier
Abstract
S-nitrosylation is a widespread modifi cation of proteins. In plants, most information available to date
regarding this modifi cation was obtained using nitric oxide donors and concerned the proteins but not
the identifi cation of cysteine residues specifi cally modifi ed in the proteins or their quantifi cation. Here, we
describe a method for the identifi cation of endogenously nitrosylated cysteines in Arabidopsis and, simul-
taneously, the measurement of relative change in their abundance within binary comparisons.
Key words
Protein S-nitrosylation, Biotin-switch, Isotope-coded-affi nity tag, Nitrosothiols,
Quantitation
Abbreviations
CID Collision-induced dissociation
DMF Dimethylformamide
EDTA Ethylenediaminetetraacetic acid
ESI-MS/MS Electrospray ionization-tandem mass spectrometry
FDR
False discovery rate
HPLC
High-performance liquid chromatography
ICAT
Isotope-coded-affi nity tag
MMTS
Methyl methanethiosulfonate
Q-TOF
Quadrupole-time of fl ight
SDS
Sodium dodecyl sulfate
Th
Thomson is a unit of mass-to-charge ratio
