Biology Reference
In-Depth Information
the sample exposed to Trx and the control (−Trx), respectively.
The two samples are mixed, digested by trypsin and the ICAT
labeled peptides are enriched by avidin chromatography. Finally,
peptides are analyzed by LC-MS/MS. ICAT
H
/ICAT
L
peptide
ratios of 1 are expected for non-target disulfide bonds that have
remained intact until the TCEP reduction. In contrast, ICAT
H
/
ICAT
L
ratios >1 are expected for peptides containing cysteines
from disulfide bonds reduced by Trx that has been blocked by
IAM and thus are not available for ICAT
L
-labeling.
2
Materials (
See
Note 1)
2.1
Trx Treatment
Trx incubation buffer: 50 mM Tris/HCl, pH 8.0, 0.2 mM EDTA,
0.72 mM NADPH (
see
Note 2
).
Slide-A-Lyzer dialysis cassettes 3.5K MWCO (Thermo Scientific
P/N 66333).
Surfactant: 1 % sodium dodecyl sulfate (SDS).
Reducing agent: 50 mM TCEP (
see
Note 3
).
Cleavable ICAT reagent (Applied Biosystems P/N 4339038).
Store at −20 °C.
N
-acetylcysteine (in 500 mM Tris/HCl pH 8.0).
Sequencing grade modified trypsin (Promega P/N V5111). Store
at −20 °C.
2.2 ICAT Labeling
and Trypsin Digestion
Cation exchange cartridge 200 μL (Applied Biosystems P/N
4326695).
Avidin affinity cartridge 200 μL (Applied Biosystems P/N
4326694).
Cartridge holder (Applied Biosystems P/N 4326688).
Needle port adaptor (Applied Biosystems P/N 4326689).
Outlet tubing kit (Applied Biosystems P/N 4326690).
Cation exchange loading buffer: 10 mM KH
2
PO
4
, 25 % acetoni-
trile, pH 3.0.
Cation exchange elution buffer: 10 mM KH
2
PO
4
, 350 mM KCl,
25 % acetonitrile, pH 3.0.
Cation exchange cleaning buffer: 10 mM KH
2
PO
4
, 1 M KCl, 25 %
acetonitrile pH 3.0.
Cation exchange storage buffer: 10 mM KH
2
PO
4
, 25 % acetonitrile
pH 3.0, 0.1 % NaN
3
.
Avidin affinity loading buffer: 20 mM NaH
2
PO
4
, 300 mM NaCl,
pH 7.2.
2.3 Purification of
ICAT-Labeled Peptides
