Supplementary Material
The Q and T parameters were varied in an attempt to maximize the ion fragmentation/trapping
efficiency, and, therefore, increase the number of identified/quantified proteins. Four
experiments, involving MCF-7 proteins labeled with iTRAQ reagents (114, 115, 116 and 117)
and mixed in a ratio of 1:1:1:1, were conducted with Q and T each set at two different levels (a
total of four sets of conditions). Two injections were performed for each experiment, and the
number of identified and quantified proteins was determined. The reproducibility in terms of
number of identified proteins was in agreement with other experiments that involved data
dependent analysis (see discussion in future sections of this manuscript). The results are
summarized in the Q/T Optimization Table. Conditions 1 and 4 (Q=0.5/T=0.1 and
Q=0.7/T=0.4) enabled the identification of very few peptides and proteins and were deemed
inadequate for effective peptide fragmentation. In addition, for condition 1, iTRAQ ratios were
hardly generated, while for condition 4, were generated for only 40-50 % of the identified
proteins. Conditions 2 and 3 (Q=0.7/T=0.1 and Q=0.5/T=0.4) generated the largest number of
identified proteins (94-135). Most of the peptides/proteins were also accompanied by iTRAQ
ratios. As the Q=0.7/T=0.1 condition for this new ion dissociation method confirmed the
recommendations provided by the manufacturer, all future experiments were performed using
these parameter settings.
Q/T Optimization Table
Effect of key PQD parameters (Q, T) on the number of identified proteins in the MCF-7 cell line.
Conditions: MCF-7 cells were cultured in EMEM/insulin, labeled with iTRAQ reagents 114,
115, 116 and 117, and mixed in a ratio of 1:1:1:1. Reporter ion 114 was used as a reference. The
protein concentration in the final sample subjected to LC-MS/MS was ~0.4 μg/μL. LC injection
volumes were 8 μL. Proteins that were counted in the analysis had P<0.001, and were matched
by peptides that passed the Xcorr vs. charge state filter (1.9, 2.2, 3.8).
PQD parameters
Q/T (ms)
1. Q = 0.5/T = 0.1
2. Q = 0.5/T = 0.4
3. Q = 0.7/T = 0.1
4. Q = 0.7/T = 0.4
# Identified proteins
Run 1
Run 2
5
6
111
125
135
94
52
39
1
Global iTRAQ ratios
Run 1
Run 2
1:0.37:ND:0.60
1:1.18:0.40:1.37
1:1.08:0.70:1.56
1:1.16:0.81:1.70
1:1.15:0.72:1.75
1:1.21:0.75:1.70
1:1.58:0.82:2.09
1:1.70:1.09:2.14
Collision Energy Settings
We present below the results of an experiment that involved the analysis of two cell states of
MCF-7 cells. Two replicates of each cell state were labeled with two iTRAQ reagents each (cell
state 1 with reagents 114 and 115, and cell state 2 with reagents 116 and 117) and mixed in a
ratio of (1:1):(1:1). The sample was analyzed by LC-MS/MS using the conditions described in
the experimental section and CE settings of 23 %, 28 %, 31 % and 35 %. All other conditions
were kept constant. In this particular experiment, the number of protein I.D.s max out at CE=31
%, while at CE=23 %, iTRAQ ratios were not measurable. The number of identified proteins
with P<0.001 and the global correction factors for each cell state are summarized in the CE
Setting Table. The optimal CE values changed in time, thus frequent optimization is probably
highly advisable.
CE Setting Table
CE setting, %
# Identified proteins
Global iTRAQ ratios
23
10
iTRAQ ratios could not be generated
28
85
1 : 1.06 : 0.68 : 0.53
31
87
1 : 0.99 : 0.63 : 0.50
35
58
1 : 1.06 :0.73 : 0.72
2
Scatter Plot of Average Z-scores for the 255 Protein Set
Conditions: MCF-7 cells were grown in the presence of E2 and Tam using the following
conditions: (A) E2 (1 nM) and (C) E2 (10 pM)/Tam (1 μM). Two experimental replicates were
performed for each cell state. Protein digests from cell condition A were labeled with reagents
114 and 115, while protein digests from cell condition C were labeled with reagents 116 and
117. The samples were mixed in a ratio (A:A):(C:C) of (1:1):(1:1). The protein amount
consumed for each labeling reaction was 100 μg. Multiconsensus data from five LC-MS/MS
experiments were used in the analysis (8 μL injections, ~2 μg/μL protein concentration). Peptide
level filtering was performed with the Xcorr vs. charge state filter set at 1.5, 2.0, 3.0. Protein
(P<0.001) level filtering is described in the text.
Average Z-score
6.00
4.00
2.00
0.00
-2.00
-4.00
-6.00
0
25
50
75
100 125 150 175 200 225 250 275
Protein #
3
Tandem Mass Spectrum of a Relevant Peptide Belonging to Poly(rC) Binding Protein 1.
The inset represents an enlarged view of the iTRAQ reporter ions m/z region.
#6405-6405 NL: 1.20E2
+1
y4
616.5
100
Poly(rC)-binding protein 1
95
Q*QSHFAMMHGGTGFAGIDSSSPEVK* (3+)
MH+=2894.38, -10lgP=87, Xcorr=4.85, Cn=0.43
90
85
75
#6405-6405 RT:92.34-92.34 NL: 1.20E2
70
65
+1
y1
291.2
60
y2
+2
390.4 b 6
422.4
50
+2
bo 12
720.7
45
+1
40
+3
a 17
+2
y* 11
608.4
+1
bo 4
607.5
+2
y9
553.3
30
25
+2
y* 6
386.8
20
+3
10
+1
b6
y7
877.5
625.4
35
+1
843.5
b5
+1
772.5
y5
703.5
90
85
80
75
116
70
65
60
55
115
50
45
40
35
30
117
114
25
20
15
10
5
0
110
111
112
113
114
115
116
117
118
m/z
119
120
121
122
123
124
125
+1
m/z
+1
+2
+1
+1
95
b7
974.7
y* 18
952.8
y3
b1
174.2 273.2
a1
245.3
100
Relative Abundance
+1
55
15
Relative Abundance
Relative Abundance
Relative Abundance
80
y* 8
975.5
+1
y9
+1
b8
+2
1105.7 1105.7
+1
y 10
a 24
1288.2
1162.6
+3
b7
325.5
+1
+2
+1
b 20
1096.9
5
+1
y 13
b 13
1438.0 1514.8
y* 16
1636.1
0
200
400
600
800
1000
m/z
m/z
4
1200
1400
1600
1800
126