Biology Reference
In-Depth Information
2.13 HPLC Tandem
Mass Spectrometry
1. Nano-HPLC system.
2. Mass spectrometer equipped with a nano-electrospray ion
source (
see
Note 10
).
3. FA solution: 0.1 % in water.
4. Reverse-phase (RP) chromatography buffer A (RPB-A):
0.1 % FA.
5. RP chromatography buffer B (RPB-B): 0.1 % FA (v/v) in 90 %
(v/v) acetonitrile (ACN).
6. Reverse-phase chromatography trap column: Zorbax 300SB-
C18, 5 mm × 0.3 mm, 5
m particle size (Agilent Technologies).
7. RP chromatography analytical column: Zorbax 300SB-C18
analytical column, 150 mm × 75
μ
μ
m, 3.5
μ
m particle size
(Agilent Technologies).
8. Search engines for LC-MS/MS protein identifi cation
(
see
Note 10
).
1. Spot picker (Gel Company).
2. Desalting cartridges Zip Tips C18 (Agilent Technologies).
2.14 MALDI-TOF
Mass Spectrometry
3. Peptide calibration standard (Bruker Daltonics) that consists
of a combination of peptides that provides a good calibration
across a typical mass range between 1,000 and 3,500 Da.
4. 0.1 % Trifl uoroacetic acid (TFA).
5.
-cyano-4-hydroxycinnamic acid
(CHCA, Sigma-Aldrich) in 70 % ACN.
6. AnchorChip MALDI target (Bruker Daltonics).
7. Autofl ex mass spectrometer (Bruker Daltonics).
8. MASCOT: Search engine for peptide mass fi ngerprint protein
identifi cation.
Matrix solution: 4.7 mg/mL
α
1. Peaks Studio v4.5 SP2 (
www.bioinformaticssolutions.com
)
.
2.15 Peptide De
Novo Sequencing
3
Methods
3.1 Plant Cell
Cultures and Culture
Conditions
(Establishment of
Callus Cultures and
Suspension- Cultured
Cells)
We describe here how a plant cell culture (
see
Note 11
) is initi-
ated and what the culture conditions are, in order to use the
extracellular medium for analyzing the extracellular proteome
(secretome).
1. Disinfect immature fruits from
Vitis vinifera
by immersing fi rst
in 70 % ethanol solution for 1 min and then into 7 % (w/v)
calcium hypochlorite solution with 0.1 % Tween 20 for 15 min
(
see
Note 12
).
