Biology Reference
In-Depth Information
(c) Sample binding: Pipette solution in the center of the well
all of the volume in the tubes, including all of the insolu-
ble parts that may remain in the solution. Confi rm visually
that the membrane absorbs the peptides. Incubate for
3 min at room temperature.
(d) Sample desalting: Add 400
μ
L of
dd
H
2
O to each well
(repeat fi ve times)
(e) Sample recovery: Replace the under well tray with a clean
one and elute peptides by adding 200
L of MeOH (repeat
once). Transfer the MeOH-peptides to low binding tubes
and evaporate completely in speedvac.
(f) Store the tubes at −20 °C until LC-MS/MS analysis
μ
3.5 Protein
Identifi cation
and Quantitation
1. Resuspend peptide pellets in 15
L of PS buffer. The amount
of buffer should be proportional to the initial amount of
peptides (
see
Note 10)
.
2. Incubate for 10 min on ice. Resuspend the pellet by pipetting
and sonicate for 3 min in a low intensity ultrasound bath.
Incubate 5 min at room temperature.
3. Spin down at >20,000 ×
g
for 10 min and at 4 °C to remove
insolubles.
4. Transfer supernatant to a LC microvial, take special care in not
disturbing the pellet or taking any clump.
μ
3.5.1 Peptide
Preparation
1. LC settings: 10
L were loaded
per injection onto a one-dimensional (1D) nano-fl ow LC-MS/
MS system (Eksigent, Germany) equipped with an in line pre-
microfi lter (Scivex, USA). Peptides were eluted using a mono-
lithic C18 column Chromolith RP-18r (Merck, Germany) of
15 cm length and 0.1 mm internal diameter during a 90 min
gradient from 5 to 40 % B with a controlled fl ow rate of 400 nL
per minute. LC was coupled to MS using a ESI source. Mobile
phase A: 0.1 % Formic Acid; Mobile phase B: 90 % Acetonitrile,
0.1 % Formic Acid (
see
Note 11
).
2. MS settings: MS analysis was performed on an Orbitrap LTQ
XL mass spectrometer (Thermo, Germany). Specifi c tune set-
tings for the MS were set as follows: spray voltage was set to
1.8 kV using a 30
μ
g of digested peptides in 5
μ
3.5.2 LC-MS/MS
m inner diameter needle (PicoTip Emitter;
NewObjective, USA); temperature of the heated transfer
capillary was set to 180 °C. FTMS was operated as follows:
fullscan mode, centroid, resolution of 30,000, covering the
range 300-1,800
m
/
z
, and Cyclomethicone was used as lock
mass. Each full MS scan was followed by ten dependent MS/
MS scans, in which the ten most abundant peptide molecular
ions were dynamically selected, with a dynamic exclusion window
set to 90 s and exclusion list set to 500. Dependent fragmentations
were performed in CID mode, with a normalized collision
μ
