Biology Reference
In-Depth Information
freshly prepared cleaving reagent to each sample tube. Vortex
to mix and then centrifuge for a few seconds to bring all solu-
tion to the bottom of the tube. Incubate for 2 h at 37 °C.
Centrifuge the tube for a few seconds to bring all solution to
the bottom of the tube. Evaporate the sample to dryness in a
centrifugal vacuum concentrator (~30 to 60 min). Resuspend
the peptides in 0.1 % formic acid and desalt them on C18
desalting microcolumns (
see
Note 5
) prior to MS analysis.
1. Peptides were resolved on an HPLC coupled to a mass spec-
trometer using C18 PepMap100 phase for pre-column and
column (75
3.3 Mass
Spectrometry and
Data Analysis
m diameter, 250 mm length) and a 2-27 % sol-
vent B (90 % acetonitrile, 0.1 % formic acid) linear gradient,
eluted into a Q-TOF mass spectrometer and fragmented by
CID [
8
,
9
] (
see
Note 6
). For peptide identifi cation, raw data
were processed using the DataAnalysis software (Bruker) and
the UniProtKB database was searched through the Mascot
(Matrix Science, v.2.2.04) engine, both in target and decoy
mode, using 20 ppm and 0.05 Da mass tolerance for peptides
and fragments respectively, methionine oxidation and ICAT
light or heavy as variable modifi cation and allowing one
missed cleavage. Signifi cant matches (
p
0.05) were selected
according to Mascot score. The FDR within a replicate was
below 2 %, with an average lower than 1 % over all experi-
ments [
10
,
11
] (
see
Note 11
). For quantitative analysis,
extracted ion chromatograms were calculated (DataAnalysis
software) and processed (Warp-LC software, Bruker) to
determine the ratios of intensity of light and heavy ICAT
labeled peptides (Fig.
2
). Ratio data were normalized to the
mean of all ratios within each replicate and averaged for each
peptide across replicates.
μ
3.4 Example
of Results
Saline stress has been applied on
Arabidopsis
suspension cells.
10 days old cells were treated with 100 mM NaCl (or water as
control) 30 min prior to be harvested. Three replicates have
been made. 123 peptides were quantifi ed with an average stan-
dard deviation of 10 %. Three peptides were found over-nitro-
sylated (left side of the sigmoid, log ratio >2.5) and 14 showed
a de-nitrosylation (right side of the sigmoid, ratio
0.25)
(Figs.
2
,
3
, and
4
). Ratios close to 1 could represent constitutive
nitrosylation because related peptides are labeled in both condi-
tions (treated and control) and at the same level. This would
implicate a fairly stable and widespread basic nitrosylation level
in many proteins. Additional controls (
see
Note 9
) show that
this labeling is likely induced by an incomplete blocking of free
SH by MMTS.
≤
