Biology Reference
In-Depth Information
3.5 Phosphopeptide
Enrichment
by HAMMOC
1. Dissolve desalted peptides in 200
l Solution C.
2. Place the HAMMOC StageTip on a micro-centrifuge tube
using the spin adaptor.
3. Conditioning of the HAMMOC StageTip with Solution A.
Load 80
μ
l Solution A onto the HAMMOC StageTip. Spin
the HAMMOC StageTip to force Solution A through.
μ
4. Conditioning of the HAMMOC StageTip with Solution C.
Load 80
l Solution C onto the HAMMOC StageTip. Spin
the HAMMOC StageTip to force Solution C through.
μ
5. Repeat
step 4
.
6. Replace the waste micro-centrifuge tube with a new tube.
Load the dissolved sample onto the conditioned HAMMOC
StageTip. Spin the HAMMOC StageTip to force the sample
through.
7. Collect the fl ow-through and load onto the same HAMMOC
StageTip again. Spin the HAMMOC StageTip to force the
sample through.
8. Washing of the HAMMOC StageTip with Solution C. Load
80
l Solution C onto the HAMMOC StageTip. Spin the
HAMMOC StageTip to force Solution C through.
μ
9. Washing the HAMMOC StageTip with Solution A. Load
80
l Solution A onto the HAMMOC StageTip. Spin the
HAMMOC StageTip to force Solution A through. This step is
necessary to wash out non-phosphopeptides which may be
bound to the C8 disk.
10. Add 100
μ
l 20 % phosphate to a 1.5 ml micro-centrifuge tube
into which phosphopeptides will be eluted.
11. Elution of the phosphopeptides from the TiO
2
beads with
Elution buffer 1 (5 % Ammonia). Replace the waste micro-
centrifuge tube with a tube containing 20 % phosphate. Load
50
μ
l Elution buffer 1 on the HAMMOC StageTip. Spin the
HAMMOC StageTip to elute phosphopeptides into the tube.
Phosphopeptides are unstable in elution buffers under basic
conditions, and eluents have to be acidifi ed immediately.
12. Elution of remaining phosphopeptides from TiO
2
beads with
Elution buffer 2 (5 % Piperidine) (
see
Note 17
). Load 50
μ
l
Elution buffer 2 onto the HAMMOC StageTip. Spin the
HAMMOC StageTip to elute phosphopeptides into the tube.
μ
1. Prepare a C18 200
μ
l StageTip.
3.6 Desalt the
Phosphopeptides with
C18 200
2. Place the C18 200
l StageTip on a 15 ml micro-centrifuge
tube using the spin adaptor.
3. Conditioning of the C18 200
μ
μ
l StageTip
μ
l StageTip with Solution A.
Load 40
μ
l Solution A onto the C18 200
μ
l StageTip. Spin the
C18 200
μ
l StageTip to force Solution A through.
