Biology Reference
In-Depth Information
25. Intra-gel spot analysis: DeCyder v6.5.11 (GE Healthcare).
26. Evaporator: Speed vacuum (Heto, Copenhagen, Denmark).
27. In-gel digestion buffer: 50 mM NH
4
HCO
3
(pH 7.8).
28. Trypsin stock solution: Sequencing grade modifi ed trypsin
(Promega, Madison, WI, USA), V5111, 5 vials (20
μ
g each),
18,100 U/mg. Dissolve 20
μ
g of one vial in 1 mL of 50 mM
NH
4
HCO
3
.
29. Spot destaining buffer: 40% (v/v) 50 mM NH
4
HCO
3
in
acetonitrile.
2.3. Analysis by
NanoLC-MS/MS
1.
NanoLC-MS/MS system (Agilent).
2.
LTQ mass spectrometer (Thermo Fisher Scientifi c, San Jose,
CA, USA).
3.
Capillary column: 150 × 0.075 mm (Proxeon/Thermo Fisher
Scientifi c).
4.
Slurry matrix: 5
m, 100-Å pore-size Magic C18 stationary
phase (Michrom Bioresources, Auburn, CA).
μ
5.
Mobile phase A: 0.1% formic acid in distilled water.
6.
Mobile phase B: 0.1% formic acid in acetonitrile.
7.
Peak list generation: Xcalibur 2.1 (Thermo Fisher Scientifi c).
8.
Peptide data searching: Mascot 2.1.03. (Matrix Science,
London, UK) using the NCBInr 06/08/2010 database.
9.
MS/MS raw data conversion: BioWorks software (version 3.2,
Thermo Fisher Scientifi c).
3. Methods
3.1. Collection
and Preparation
of Clinical Samples
1.
According to the standard protocol for reference plasma sample
collection recommended by the Human Proteome Organization
(HUPO) (
1
)
,
collect the blood of healthy donors and HCC
patients into K
2
EDTA tubes, and leave at room temperature
for 30 min. Then centrifuge the tubes at 2,400 ×
g
for 15 min
to remove red blood cells and cellular particles. Transfer the
upper liquid phase (plasma) into cryovials and store at −85°C
until use (see Note 3).
2.
Dilute 500
L of human plasma with 2 mL of MARS Buffer A,
and add 100
μ
μ
L of protease inhibitor cocktail solution. Inject
100
L of the diluted plasma into the Agilent HP1100 LC
system equipped with a MARS affi nity column at a fl ow rate of
0.25 mL/min. Collect fl ow-through fractions, precipitate by
addition of 50% TCA solution, and then store the pellet at
−20°C overnight (see Note 4).
μ
Search WWH ::
Custom Search