Biology Reference
In-Depth Information
4. Conditioning of the C18 200
μ
l StageTip with Solution B.
Load 40
μ
l Solution B onto the C18 200
μ
l StageTip. Spin the
l StageTip to force Solution B through.
5. Load the acidifi ed phosphopeptide enriched sample onto the
conditioned C18 200
C18 200
μ
l
StageTip to bind peptides to the C18 disks and force the rest
through.
6. Washing of the C18 200
μ
l StageTip. Spin the C18 200
μ
μ
l StageTip with Solution B. Load
40
μ
l Solution B on the C18 200
μ
l StageTip. Spin the C18
l StageTip to force Solution B through.
7. Elution of the bound peptides from the C18 disks with
Solution A. Replace the waste conical tube with a new tube.
Load 40
200
μ
μ
l Solution A on the C18 200
μ
l StageTip. Spin the
C18 200
μ
l StageTip to elute peptides into the clean tube (
see
Note 18
).
8. Dry the eluent in a SpeedVac. Dried peptide sample can be
stored at −20 °C.
9. Dissolve the desalted peptides in buffer for LC-MS/MS
analysis.
4
Notes
1. We add 1 ml buffer to the tube and vortex immediately.
Phosphatase inhibitors may not be required in the denaturing
extraction buffer. If alternative extraction buffers without
denaturants are used, don't forget to add phosphatase
inhibitors.
2. The specifi city of TiO
2
beads against phosphopeptides is
reduced by water absorption when it is kept without desicca-
tion. Specifi city can be recovered by heating the beads in a
drying oven at 130 °C for 30 min.
3. Other methods such as fi lter-aided sample preparation (FASP)
and phase-transfer surfactant (PTS) protocol may help recover
more membrane proteins [
19
,
20
].
4. We usually prepare 2 ml micro-centrifuge tubes suitable for
bead mill apparatus with 4 zirconia beads (3 mm diameter) for
each tube, and harvest ca. 200 mg plant materials directly into
the tubes. Samples are frozen in liquid nitrogen and can be
kept at −80 °C.
5. Not necessary to be a precise ratio.
6. For a sample intended for trypsin digestion, we use 0.5
μ
g
LysC for every 50
g protein. It is not necessary to digest with
LysC prior to trypsin. However, LysC digestion under dena-
μ
