9. Load the desalted peptides onto the prepared IMAC tip by
centrifugation at 300 Â g at 25
C for 2 min.
10. Wash the IMAC tip with 100 μL of 80% ACN and 0.1% TFA to
remove unbound nonphosphopeptides by centrifugation at
300 Â g at 25
C for 2 min (see Note 10).
11. Wash the IMAC tip with 100 μL of 80% ACN and 1% TFA to
remove unbound nonphosphopeptides by centrifugation at
300 Â g at 25
C for 2 min (see Note 10).
12. Wash the IMAC tip with 100 μL of 1% acetic acid to remove
unbound nonphosphopeptides by centrifugation at 300 Â g at
25
C for 2 min (see Note 11).
13. Elute the bound phosphopeptides with 200 μL of 200 mM
NH 4 H 2 PO 4 by centrifugation at 200 Â g at 25
C for 2 min.
Repeat this step one more time.
14. Transfer the eluted phosphopeptides to the C18 StageTip by
centrifugation at 1000 Â g at 25
C for 4 min.
3.7 Peptide
Desalting by C18
StageTip for Small
Amounts of Samples
1. Use a 16-Gauge needle to punch a hole on the C18
membrane disk.
2. Use a 20-Gauge needle to help insert the punched membrane
disk into the D200 tip (the loading capacity of ~20 μg) (see
Note 12).
3. Activate the C18 StageTip with 40 μL of MeOH and then
buffer B.
4. Equilibrate the C18 StageTip with 40 μL of buffer A.
5. Load the enriched phosphopeptides onto the C18 StageTip.
6. Wash the C18 StageTip with 40 μL of buffer A two times.
7. Elute phosphopeptides directly into the sample vial with 40 μL
of buffer B.
8. Dry down the phosphopeptides with SpeedVac and then store
at À80
C until LC-MS analysis.
9. Reconstitute the phosphopeptides with 2% ACN and 0.1% FA
for LC-MS analysis.
3.8 LC-MS Setup
Example with Waters nanoACQUITY LC and Orbitrap Fusion
Lumos Tribrid mass spectrometer.
A column was packed in-house using 20-cm-long fused silica
with 75 μm inner diameter and packed with 1.9-μm ReproSil C18
resin.
1. Load 5 μL of the sample (reconstituted in 4% ACN and 0.1%
FA) on the column and separate through a gradient (2–6%
solvent B for 1 min, 6–30% solvent B for 84 min, 30–60%
solvent B for 9 min, 60–90% solvent B for 1 min, and finally
90% solvent B for 5 min) at a flow rate of 200 nL/min.
254
Chia-Feng Tsai et al.
centrifugation at 300 Â g at 25
C for 2 min.
10. Wash the IMAC tip with 100 μL of 80% ACN and 0.1% TFA to
remove unbound nonphosphopeptides by centrifugation at
300 Â g at 25
C for 2 min (see Note 10).
11. Wash the IMAC tip with 100 μL of 80% ACN and 1% TFA to
remove unbound nonphosphopeptides by centrifugation at
300 Â g at 25
C for 2 min (see Note 10).
12. Wash the IMAC tip with 100 μL of 1% acetic acid to remove
unbound nonphosphopeptides by centrifugation at 300 Â g at
25
C for 2 min (see Note 11).
13. Elute the bound phosphopeptides with 200 μL of 200 mM
NH 4 H 2 PO 4 by centrifugation at 200 Â g at 25
C for 2 min.
Repeat this step one more time.
14. Transfer the eluted phosphopeptides to the C18 StageTip by
centrifugation at 1000 Â g at 25
C for 4 min.
3.7 Peptide
Desalting by C18
StageTip for Small
Amounts of Samples
1. Use a 16-Gauge needle to punch a hole on the C18
membrane disk.
2. Use a 20-Gauge needle to help insert the punched membrane
disk into the D200 tip (the loading capacity of ~20 μg) (see
Note 12).
3. Activate the C18 StageTip with 40 μL of MeOH and then
buffer B.
4. Equilibrate the C18 StageTip with 40 μL of buffer A.
5. Load the enriched phosphopeptides onto the C18 StageTip.
6. Wash the C18 StageTip with 40 μL of buffer A two times.
7. Elute phosphopeptides directly into the sample vial with 40 μL
of buffer B.
8. Dry down the phosphopeptides with SpeedVac and then store
at À80
C until LC-MS analysis.
9. Reconstitute the phosphopeptides with 2% ACN and 0.1% FA
for LC-MS analysis.
3.8 LC-MS Setup
Example with Waters nanoACQUITY LC and Orbitrap Fusion
Lumos Tribrid mass spectrometer.
A column was packed in-house using 20-cm-long fused silica
with 75 μm inner diameter and packed with 1.9-μm ReproSil C18
resin.
1. Load 5 μL of the sample (reconstituted in 4% ACN and 0.1%
FA) on the column and separate through a gradient (2–6%
solvent B for 1 min, 6–30% solvent B for 84 min, 30–60%
solvent B for 9 min, 60–90% solvent B for 1 min, and finally
90% solvent B for 5 min) at a flow rate of 200 nL/min.
254
Chia-Feng Tsai et al.