Biology Reference
In-Depth Information
1
Introduction
S-nitrosylation, the modifi cation of the thiol group of cysteines by
nitric oxide (NO) constitutes a wide-spread post-translational
modifi cation of crucial importance in a large array of processes,
including in plants [
1
]. At the protein level, the “biotin-switch”
method constituted a breakdown for the identifi cation of nitrosyl-
ated proteins [
2
]. This method consists in blocking non-nitrosylated
cysteines, and then reducing nitrosylated ones, prior to conjuga-
tion with a thiol reagent bound to biotin, thus allowing the selec-
tion of proteins containing formerly nitrosylated cysteines by
avidin-biotin interaction. Very recently, at the peptide and amino
acid level, the use of a thiol-specifi c isotope-coded affi nity tag
(ICAT) was reported to permit comparative quantifi cation of dif-
ferently nitrosylated peptides [
3
]. In both cases, however, nitrosyl-
ation was favored using a NO donor.
In the plant area, very similar progresses were noticed. Notably,
the use of the biotin-switch allowed fi rst to determine sets of nitro-
sylated proteins and then to identify proteins differentially nitrosyl-
ated in response to various stresses [
2
,
4
-
6
]. However, as for other
organisms, NO donors were used, thus preventing to decipher
naturally occurring nitrosylation events, and all comparative analy-
sis were based on quantifi cation of nitrosylated proteins, without
information about the concerned nitrosocysteines. Moreover, no
approach aiming at large-scale identifi cation of nitrosylated resi-
dues was published to date.
We detail here (Fig.
1
) a combination of the biotin-switch
method and ICAT labeling that was proven [
7
] to allow, for
Arabidopsis
proteins, the large-scale identifi cation of endogenous
nitrosothiols, in the absence of NO donor.
2
Materials
Prepare all solutions using ultrapure water (prepared by purifying
deionized water to attain a resistivity of 18.2 M
cm at 25 °C) and
analytical grade reagents. Prepare and store all reagents at 4 °C
(unless indicated otherwise). Diligently follow all waste disposal
regulations when disposing waste materials. Do not add sodium
azide to the reagents. Wear appropriate personal protective equip-
ment (safety glasses, gloves, protective clothing) when handling
chemicals such as MMTS, DMF, SDS, NaOH, ICAT reagents and
buffers and use them only with adequate ventilation (e.g., fume
hood). All the experiments from the cell grinding to the ICAT
labeling are done in the darkness to protect nitrosothiols from
light. All the 1.5 ml microcentrifuge tubes used in this experiment
are LoBind Eppendorf tubes (Eppendorf 022431081) reducing
adsorption to the tube walls.
Ω
