Biology Reference
In-Depth Information
Fig. 1
Structure of acid-cleavable ICAT reagents. The reagent contains four different functionalities: (1) a iodoacetamide
group reacting with reduced thiol groups (−SH), (2) a tag with nine isotope-coded carbon atoms (x =
13
C
(ICAT “heavy”); x =
12
C(ICAT “light”), (3) a biotin tag that serves as an affinity tag for avidin chromatography to
enrich labeled peptides and (4) an acid-cleavable linker. Reproduced from ref.
3
with permission from American
Chemical Society
specific target disulfide bonds and the extent of their reduction by
Trx is unknown. This information is important for an in-depth
understanding of the role of Trx in various metabolic pathways. We
addressed this issue by developing a quantitative differential thiol
labeling procedure using isotope-coded affinity tag (ICAT) reagents
[
3
,
4
]. The ICAT reagents contain a thiol-reactive iodoacetamide
(IAM) group and isotope coded linkers available in “light” (ICAT
L
)
and “heavy” (ICAT
H
) forms labeled with
12
C and
13
C stable carbon
isotopes, respectively (Fig.
1
). The relative abundance of cysteine
containing tryptic peptides in two samples reacted with ICAT
L
and
ICAT
H
, respectively, can thus be determined by measuring ratios of
labeled peptides analyzed by LC-MS/MS. The ICAT reagent also
contains a biotin tag for selective enrichment of labeled peptides by
avidin affinity chromatography.
ICAT was initially developed in a deuterated form as the first
generation of reagents for global quantitative proteomics using
stable isotope labeling [
14
].
ICAT has since been surpassed by alternative reagents (e.g.,
iTRAQ) that are better suited for general quantitative proteome
analysis. Nevertheless, ICAT reagents are still very attractive in the
niche of thiol-specific proteomics and the reagents have been suc-
cessfully applied in various biological systems [
15
-
22
]. Since ICAT
reacts with reduced thiol groups and not with oxidized forms (e.g.,
disulfide bonded cysteines) the reagent is well suited to monitor
thiol oxidoreduction processes. Here, ICAT reagents are applied
for Trx target identification in a multistep, differential labeling
procedure (Fig. 2): (1) free thiols in protein extracts are blocked
with IAM, (2) parallel samples incubated in the presence or absence
of Trx are added IAM to quench Trx and block reduced thiols, (3)
remaining protein thiols are fully reduced with tris(2-carboxyethyl)
phosphine (TCEP) followed by addition of ICAT
L
and ICAT
H
to
