Biology Reference
In-Depth Information
2. SCX chromatography buffer B (SCX-B): 10 mM KH 2 PO 4 ,
0.5 M KCl, 20 % ACN, pH 2.7 in HPLC-grade water.
3. SCX chromatography buffer C (SCX-C): 10 mM KH 2 PO 4 ,
1 M KCl, 20 % ACN, pH 2.7 in HPLC-grade water.
4. SCX chromatography column: Mono S PC 1.6/5 sulfonate
column, 1.6 × 50 mm, 10
m beads (GE Healthcare).
5. Desalting activation solution: 50 % ACN.
6. Desalting sample buffer, equilibration, and wash buffer: 0.5 %
trifl uoroacetic acid (TFA) in 5 % ACN.
μ
7. Desalting elution buffer: 0.1 % TFA in 70 % ACN.
2.3 Sample Analysis
by LC-MS/MS
1. Reverse-phase (RP) chromatography buffer A (RPB-A): 0.1 %
formic acid (FA) (v/v) and 5 % ACN in water.
2. RP chromatography buffer B (RPB-B): 0.1 % FA in ACN.
3. RP chromatography trap column: Zorbax 300SB-C 18 cartridges,
5 × 0.3 mm, 5
m particle size (Agilent Technologies).
4. RP chromatography analytical column: Zorbax 300SB RP C 18
column, 75
μ
μ
m × 150 mm, 3.5
μ
m particle size (Agilent
Technologies).
3
Methods
The technique and procedure detailed have been used in our group
for the proteomic analysis of grapevine ( Vitis vinifera L) berries
[ 9 ]. First, the tissue was extensively cleaned up to remove polyphe-
nols and lipids, and then proteins were extracted with phenol and
precipitated with ammonium acetate-methanol.
1. Prepare a protein sample free of contaminants and impurities
avoiding potential interfering substances with iTRAQ protocol.
These interfering substances are thiols (i.e., DTT, mercaptoeth-
anol), high amounts of detergents and denaturants, proteases,
and primary amines (e.g., Tris buffers). In such case, dilute your
sample adding at least six to ten volumes of chilled acetone and
incubate overnight at −20 °C. After centrifugation at maxi-
mum speed for 10 min using a benchtop centrifuge, decant
acetone and centrifuge for an additional 2 min, using a pipette
to remove any remaining acetone ( see Note 2 ).
3.1 Sample
Preparation
and Labeling
2. The protein pellet without drying is immediately resuspended
by addition in sequential order of fresh saturated urea/0.1 M
TEAB 1/1 (v/v) and SDS 2 % (w/v) to a fi nal concentration
of 0.2 %. Solubilize completely the protein sample if necessary
by incubation overnight at 4 °C or alternatively by sonication
( see Note 3 ).
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