Biology Reference
In-Depth Information
7,00
6,00
5,00
4,00
3,00
2,00
1,00
0,00
0
20
40
60
80
100
120
140
Accessions identified
-1,00
Fig.
4
Quantitation of nitrosothiols labeled with ICAT reagents in
Arabidopsis
cells treated with NaCl during
30 min. Three replicates have been made and only peptides present in all three replicates were quantifi ed.
Ratio mentioned in the fi gure represent treated cells/control. Every single point in the fi gure represents an
accession and the standard deviation is indicated for each accession
4
Notes
1. Cells are quite sensitive to any changes and we do prefer to
stick to the same medium provider.
2. This type of blender is recommended for its high effi ciency on
frozen material.
3. 2D Quant Kit is able to deal with high concentration of SDS
and reductant.
4. We tried different fi lter devices and Amicon Ultra gave us the
best compromise yield/speed of the concentration process.
5. C18 desalting microcolumns can be used at different times to
get rid of impurities in peptide samples but some reagents like
SDS are incompatible with C18. Depending of the sample vol-
ume, Zip-Tip pipette tips from Millipore (ZTC18M096) can
be used (vol <150
μ
L) or Sep-Pak from Waters WAT036820
(bigger volumes).
6. The procedure described here used a Q-TOF mass spectrom-
eter (Maxis, Bruker), coupled to an Ultimate 3000 HPLC
(Dionex), and corresponding Bruker software (DataAnalysis,
Warp-LC).
7. The 50 mL centrifuge tubes are acetone resistant and can be
spun at 6,000 ×
g
. 45 mL of cold acetone are added to 5 mL of
sample in each 50 mL centrifuge tube.
8. Depending of the sample concentration, two fi lter devices can
be used (Amicon Ultra-4 or -15). Moreover, when fi lter devices
