Biology Reference
In-Depth Information
a
c
H
N
C
C
His
110.0713
Low mass region of
the MS/MS
spectrum
C
PEPTIDE
reporter ions
N
C
N
1.0
0.8
0.6
0.4
0.2
0.0
O
N
O
N
117.1143
Phe
120.0806
114.1107
115.1078
13
C
13
C
18
O
114
31
NHS
116.1111
111.0746
110
112
114
116
118
120
m/z
13
C
2
NHS
18
O
115
30
MS/MS spectrum
13
C
2
/
15
N
13
C
29
NHS
116
y
1
5
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
549.3153
110.0713
precursor ion
13
C
3
/
15
N
28
NHS
117
b
1+
5
b
1
4
y
2
6
b
2+
6
627.3591
514.2753
343.6908
438.5802
382.7134
y
1
6
117.1143
Isotopical
combinaons
Reporter group
114-117 amu
Balance group
31-28 amu
686.3751
100
200
300
400
500
600
700
800
Isobaric tag - total mass 145
(reporter group + balance group)
m/z
b
MS/MS
fragmentaon
(
e.g.,
CID, HCD)
iTRAQ
labeled pepdes
Sample A
*
signal ion
intensies
resulng from the
addion of
pepdes bearing
four different
iTRAQ labels
Analyse
chromatographic
fracons by
RP-LC-MS/MS
Pool and
fraconate by
SCX
Sample B
Sample C
Sample D
mass /charge
Reducon (TCEP)
Alkylaon (MMTS)
Trypsin
digeson
Fig. 1
(
a
) Diagram showing the components of the iTRAQ tags. The complete molecule consists of a reporter
group (based on
N
-methylpiperazine) (shadowed in
green
), a mass balance group (carbonyl) (shadowed in
blue
), and an amine-specifi c peptide-reactive group (NHS ester). The overall mass of reporter and balance
components of the molecule display a constant mass of 145 amu, the reporter group ranges in mass from 114
to 117 amu while the balance groups range from 28 to 31 amu. The mass of iTRAQ labels depends on the
isotopic combination of N, C, and O atoms within the reporter group and the balance group found in each
iTRAQ label. At the
left side
of each iTRAQ tags is depicted the precise isotopic combinations corresponding to
both reporter (
green box
) and balance (
blue box
) groups. The tags form an amide linkage to amine groups
of peptides (N-terminal or
amino group of lysine) through the NHS group when reacted with a peptide.
(
b
) An overview of a typical iTRAQ workfl ow. Peptides derived from the trypsin-digested protein samples
are labeled with the iTRAQ tags, pooled, and then fractionated by strong cation exchange chromatography.
(
c
) The resulting fractions are analyzed by LC-MS/MS in which a single ion or precursor is generated for each
peptide resulting in a summed intensity from the species present on each sample; thus a mixture of four
identical peptides each labeled with one iTRAQ tag appears as a single, unresolved precursor ion in MS
(identical
m
/
z
). As example in the diagram, the precursor ion marked with an
asterisk
is isolated and subjected
to MS/MS fragmentation using CID or HCD.
Dashed lines
correspond to a zoomed view showing the four
reporter ions resolved and detected as fragment ions with
m
/
z
values ranging from 114 to 117 amu. All other
sequence-informative fragment ions (mainly
b
- and
y
-fragment ions, displayed in
red
and
blue
color, respec-
tively) remain isobaric after fragmentation, and their individual ion current signals (signal intensities) are also
additive. Finally, the relative concentration of the peptides is deduced from the relative intensities of the cor-
responding reporter ions. Peptides, and their derived fragments ions, labeled with the iTRAQ tags are shaded
in
gray
(114),
pink
(115),
orange
(116), and
purple
(117) color
ε
