Biology Reference
In-Depth Information
Subheading
3.2
to a 15 mL falcon tube, mix with 4 mL cation
exchange loading buffer, and slowly (approx. 1 drop/second)
inject onto the cation exchange cartridge. Inject 1 mL cation
exchange loading buffer and discard the eluent. Inject 500 μL cat-
ion exchange elution buffer and capture the eluted peptides in an
eppendorf tube. Inject 1 mL cation exchange cleaning buffer and
discard the flow through. If further samples are to be processed,
re-equilibrate the cartridge with 2 mL cation exchange loading
buffer and continue as described above, otherwise inject 2 mL cat-
ion exchange storage buffer, disassemble the cartridge holder and
store the cartridge at 4-8 °C.
3.4 Avidin Affinity
Chromatography
Assemble the avidin affinity cartridge into the cartridge holder
with the needle port adaptor and the outlet tubing according to
the manufacturer's instructions. Inject 2 mL avidin affinity elution
buffer followed by 2 mL avidin affinity loading buffer to condition
the cartridge. Mix samples from Subheading
3.3
with 500 μL avi-
din affinity loading buffer and slowly (approx. 1 drop/second)
inject onto the avidin affinity cartridge. Inject 500 μL avidin affinity
loading buffer followed by 1 mL avidin affinity washing buffer 1,
1 mL avidin affinity washing buffer 2 and 1 mL H
2
O. Inject 800 μL
avidin affinity elution buffer, where the first 50 μL eluting from the
cartridge is discarded and the remaining 750 μL containing ICAT-
labeled peptides are stored. If further samples are to be processed,
re-equilibrate the cartridge with 2 mL avidin affinity elution buffer
followed by 2 mL avidin affinity loading buffer and repeat the
procedure described above, otherwise inject 2 mL avidin affinity
storage buffer, disassemble the cartridge holder and store the car-
tridge at 4-8 °C.
3.5 ICAT Tag
Cleavage
Dry samples from Subheading
3.4
in a SpeedVac centrifuge and
incubate with cleaving reagents A and B in a 95:5 ratio (100 μL
total volume) for 2 h at 37 °C to remove the biotin tag from ICAT
labeled peptides (Fig.
1
). Dry down the samples in a SpeedVac
centrifuge.
3.6 C18 StageTip
Peptide Purification
StageTips are prepared essentially as described previously [
23
] by
mounting plugs of Empore C18 solid phase extraction disks in
Gilson P200 pipette tips (
see
Note 8
). Equilibrate the C18
StageTips by loading 10 μL C18 elution buffer followed by 10 μL
C18 loading buffer. Dissolve samples from Subheading
3.5
in
10 μL C18 loading buffer and load onto the stage tip. Wash the
StageTips at least twice with 10 μL C18 loading buffer. Finally,
elute peptides with 10 μL C18 elution buffer, capture the eluate in
a 250 μL eppendorf tube and dry in a SpeedVac centrifuge.
Redissolve samples in 10 μL C18 loading buffer and transfer to a
96-well plate for LC-MS injection.
