room temperature for 2 min. The supernatant is then carefully
removed and discarded. This step is repeated two times.
3. Digested peptides are dissolved in 200 μL resuspension buffer
(see Note 10). Dissolved peptides are centrifuged at 20,000 g
for 5 min.
4. The dissolved peptides from step 3 are mixed with equilibrated
TiO 2 beads (see Note 11) for 30 min with continuous mixing
on a shaker or a vortex mixer and centrifuged at 2500 g at room
temperature for 2 min, transfer the supernatant into a new tube
(see Note 12) and keep the TiO 2 beads pellet to continue.
5. The peptides and TiO 2 beads mixture are then washed once
with 100 μL resuspension buffer with continuous mixing on
vortex for 30 s and centrifuged at 2500 g at room temperature
for 2 min. The supernatant is then carefully removed and
discarded.
6. The peptides and TiO 2 beads mixture are then washed with
200 μL wash buffer for 2 min and centrifuged at 2500 g at
room temperature for 2 min. The supernatant is then carefully
removed and discarded. This step is repeated two times.
7. The peptides and TiO 2 beads mixture are incubated with 80 μL
elution buffer for 15 min and centrifuged at 2500 g at room
temperature for 2 min. The supernatant is then collected into a
new Eppendorf tube.
8. Step 7 is repeated two times.
9. Eluates are immediately acidified with 60 μL acidified buffer
(see Note 13).
10. Eluates samples are either desalted directly or stored in À20
C
for short time and À80
C for long time.
3.4 Peptide
Desalting Using C18
StageTips (Timing
~1.5 h)
1. Preparation of C18 StageTip has been described [14] (see Note
4). An Empore Disk C18 is placed on a flat, surface-cleaned
plastic petri dish. Two layers of small Empore Disk C18 disks
are stacked into a 200-μL pipette tip. The ready-made tip can
be then placed on a holder, and assembled with the 2 mL tube
for collecting the flow-through.
2. C18 stage tips are conditioned by adding 50 μL of solution B
and centrifuged at 1500 g at room temperature for 5 min. The
flow-through is discarded.
3. C18 stage tips are equilibrated by adding 100 μL of solution A
and centrifuged at 1500 g at room temperature for 5 min. The
flow-through is discarded. This step is repeated one time.
4. Eluates from step 10 (3.3) are loaded into equilibrated C18
stage tips and centrifuged at 1500 g at room temperature for
5 min. The flow-through is discarded.
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Lin Xi et al.
removed and discarded. This step is repeated two times.
3. Digested peptides are dissolved in 200 μL resuspension buffer
(see Note 10). Dissolved peptides are centrifuged at 20,000 g
for 5 min.
4. The dissolved peptides from step 3 are mixed with equilibrated
TiO 2 beads (see Note 11) for 30 min with continuous mixing
on a shaker or a vortex mixer and centrifuged at 2500 g at room
temperature for 2 min, transfer the supernatant into a new tube
(see Note 12) and keep the TiO 2 beads pellet to continue.
5. The peptides and TiO 2 beads mixture are then washed once
with 100 μL resuspension buffer with continuous mixing on
vortex for 30 s and centrifuged at 2500 g at room temperature
for 2 min. The supernatant is then carefully removed and
discarded.
6. The peptides and TiO 2 beads mixture are then washed with
200 μL wash buffer for 2 min and centrifuged at 2500 g at
room temperature for 2 min. The supernatant is then carefully
removed and discarded. This step is repeated two times.
7. The peptides and TiO 2 beads mixture are incubated with 80 μL
elution buffer for 15 min and centrifuged at 2500 g at room
temperature for 2 min. The supernatant is then collected into a
new Eppendorf tube.
8. Step 7 is repeated two times.
9. Eluates are immediately acidified with 60 μL acidified buffer
(see Note 13).
10. Eluates samples are either desalted directly or stored in À20
C
for short time and À80
C for long time.
3.4 Peptide
Desalting Using C18
StageTips (Timing
~1.5 h)
1. Preparation of C18 StageTip has been described [14] (see Note
4). An Empore Disk C18 is placed on a flat, surface-cleaned
plastic petri dish. Two layers of small Empore Disk C18 disks
are stacked into a 200-μL pipette tip. The ready-made tip can
be then placed on a holder, and assembled with the 2 mL tube
for collecting the flow-through.
2. C18 stage tips are conditioned by adding 50 μL of solution B
and centrifuged at 1500 g at room temperature for 5 min. The
flow-through is discarded.
3. C18 stage tips are equilibrated by adding 100 μL of solution A
and centrifuged at 1500 g at room temperature for 5 min. The
flow-through is discarded. This step is repeated one time.
4. Eluates from step 10 (3.3) are loaded into equilibrated C18
stage tips and centrifuged at 1500 g at room temperature for
5 min. The flow-through is discarded.
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Lin Xi et al.