Biology Reference
In-Depth Information
Avidin affinity elution buffer: 30 % acetonitrile, 0.4 % trifluoroace-
tic acid (TFA).
Avidin affinity washing buffer 1: 10 mM NaH
2
PO
4
, 150 mM
NaCl, pH 7.2.
Avidin affinity washing buffer 2: 50 mM NH
4
CO
3
, 20 % methanol,
pH 8.3.
Avidin affinity storage buffer: 20 mM NaH
2
PO
4
, 300 mM NaCl,
pH 7.2, 0.1 % NaN
3
.
Cleaving reagent A (Applied Biosystems P/N 4338543) (
see
Note 4
).
Cleaving reagent B (Applied Biosystems P/N 4339052) (
see
Note 5
).
Store at −20 °C.
2.4 C18 StageTip
Peptide Purification
Empore C18 Extraction Disk (3M, P/N 2215).
C18 loading buffer: 0.1 % TFA.
C18 elution buffer: 80 % acetonitrile, 0.1 % TFA.
3
Methods
3.1
Trx Treatment
Equilibrate approx. 200-400 μg) protein extract (
see
Note 6
) in
400 μL Trx incubation buffer and distribute the solution in four
eppendorf tubes (A-D; 100 μL/tube). Add 2 μL NTR (final con-
centration 2 μM) and 9.6 μL Trx (final concentration 6.5 μM) to
A and C (
see
Note 7
); add 2 μL NTR and 9.6 μL water to B and
D. After 1 h incubation at RT, add 12 μL IAM (final concentration
10 mM) to A and B and 12 μL water to C and D. Incubate the
samples for 80 min at RT in the dark, cool on ice, add 100 μL
50 mM Tris/HCl pH 8.0, and dialyze against 50 mM Tris/HCl
pH 8.0 overnight at 4 °C.
Mix samples from Subheading
3.1
(72 μL) with surfactant (8 μL)
and reducing agent (2 μL) and incubate at 99 °C for 10 min. Add,
20 μL 11.4 mM cleavable ICAT reagent (in acetonitrile) to A and
C (ICAT
L
) and B and D (ICAT
H
) and incubate 80 min at RT in
the dark, followed by quenching of excess ICAT reagent by addi-
tion of 10 μL 100 mM
N
-acetylcysteine (in 500 mM Tris/HCl
pH 8.0) and incubate for 10 min at RT. Then mix the samples
(A+B and C+D), add 45 μL 100 mM CaCl
2
and adjust the sample
volume to 438 μL with water. Finally, add 10 μL sequencing grade
modified trypsin (0.1 μg/μL) and incubate the samples at 37 °C
overnight.
3.2 ICAT Labeling
and Trypsin Digestion
3.3 Cation Exchange
Chromatography
Assemble the cation exchange cartridge into the cartridge holder
with the needle port adaptor and the outlet tubing according to
the manufacturer's instructions. Inject 2 mL cation exchange load-
ing buffer to condition the cartridge. Transfer samples from
