Robust MS Quantification Method for Phosphopeptides using 18O/16O Labeling
Claus A. Andersen, Stefano Gotta, Letizia Magnoni, Roberto Raggiaschi, Andreas
Kremer and Georg C. Terstappen
Siena Biotech SpA, Discovery Research, Via Fiorentina 1, 53100 Siena, Italy
Supporting material
Full labeling
Mixt labeling
DpSDDEEEVVHVDR
KBackground
7000
TLight
KBackground
T
4500
TLight
T
4000
TMixt
3500
THeav y
3000
S [Observed]
Background
Light
6000
TMixt
S [Observed]
1000
Spectral intensity
Mixt
THeav y
1500
Fold change = NaN [NaN NaN]
5000 AKTDHGAEIVYKpSPVVpSGDTpSPR
K
T
5000
Heavy
S [Observed]
4000
3000
Spectral intensity
2000
Spectral intensity
Direct
DLpSLEEIQKK
No labeling
Lab.Eff = 0.85 Fold change = 1.02 [-1.1 1.2]
Fold change = -Inf [NaN NaN]
1000
1000
500
0
0
S1
S2
S3 S4 S5 S6
m/z [1 Da per step]
S7
0
S0
S8
Fold change = -Inf [NaN NaN]
1000
800
S2
S4 S5 S6
S3
m/z [1 Da per step]
S7
S8
S0
S1
9000 DpSDDEEEVVHVDR
K
TLight
8000
T
TMixt
7000
T
THeavy
S [Observed]
600
400
S2
S3 S4 S5 S6
m/z [1 Da per step]
S8
3500 AKTDHGAEIVYKpSPVVpSGDTpSPR
KBackground
3000
TLight
Background
Light
TMixt
Mixt
T
Heavy
6000
S7
Fold change = NaN [NaN NaN]
Lab.Eff = 0.85 Fold change = 1.02 [-1.1 1.2]
KBackground
Spectral intensity
DLpSLEEIQKK
S1
S [Observed]
5000
4000
3000
Spectral intensity
S0
Spectral intensity
2000
1500
2000
500
Inverted
2500
2500
THeav y
S [Observed]
2000
1500
1000
2000
200
500
1000
0
0
S0
S1
S2
S3 S4 S5 S6
m/z [1 Da per step]
S7
S8
0
S0
S1
S2
S3
S4 S5 S6
m/z [1 Da per step]
S7
S8
S0
S1
S2
S3 S4 S5 S6
m/z [1 Da per step]
S7
Figure S1 The degree to which a peptide is labeled (the labeling efficiency) varies dramatically
between peptides. On the left is a special case where a peptide has been fully labeled (full
labeling). This can be immediately seen since the peptide was absent from the treated sample, by
comparing the direct and inverted experiments (label swapping). One peptide was not labeled
(No labeling) and most peptides receive a partial labeling (see Figure 2b).
17-Feb-16 3:28:01 AM
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S8
80
70
mean = 1.28
median = 1.12
stddev = 0.40
Relative occurrence
60
mean = 1.00
median = 1.00
stddev = 0.86
50
40
30
20
10
0
-6
-4
-2
0
2
4
6
Estimated fold change
Figure S2 The estimated fold change is compared between the experimental spectra (red) and the
simulated spectra (blue). The simulated spectra were generated as a symmetrical distribution
with a standard deviation twice as large as the ones observed to allow the investigation of high
fold change peptides. The experimentally observed spectra pair with a completely absent treated
peptide was excluded since its theoretical fold change was –infinite.
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2
Fold change error: average(|true - estimated|)
0.9
noise [0.01 0.06]
noise (0.06 0.10]
0.8
noise (0.10 0.15]
noise (0.15 0.20]
0.7
noise (0.20 0.24]
0.6
noise (0.24 0.29]
noise (0.29 0.34]
0.5
0.4
0.3
0.2
0.1
0
0.4
0.5
0.6
0.7
0.8
0.9
1
Estimated labeling efficiency
Figure S3 When the labeling efficiency decreases the fold change error was found to increase. For
a low noise level (noise [0.01 0.10]) we found an almost constant or slowly increasing fold
change error upon decreasing labeling efficiency, while for increased noise level the fold change
error was found to increase steadily with poorer labeling efficiency.
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1
noise [0.01 0.06]
noise (0.06 0.10]
noise (0.10 0.15]
noise (0.15 0.20]
Imposed labeling efficiency
0.9
noise (0.20 0.24]
0.8
noise (0.24 0.29]
noise (0.29 0.34]
0.7
0.6
0.5
0.4
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Estimated labeling efficiency
Figure S4 The labeling efficiency estimate is generally quite reliable, but depends on the level of
noise in the spectra. An interesting aspect is that on average it is overestimated for high labeling
efficiencies and underestimated when it is low. The black line illustrates the ideal labeling
efficiency.
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Fold change: 1.11 [1.03 1.20]
Fold change error: average(|true - estimated|)
KBackground
S3 S4 S5 S6
m/z [1 Da per step]
S8
TLight
Inverted
Inverted
TMixt
THeavy
S [Observed]
0.4
S2
S3
S4
S5
S6
S7
0.45
0.3
0.1
S1
0.2
5
0.05
0.5
0.02
S8
0.45
0.4
0.6
0.2
S0
0.1
0.
2
S7
KBackground
0.4
45
0.
0.6
0.6
0.7
0.7
0.8
0.6
0.7
0.8
0.9
1
2
1.1 11.3.
1.4
0.04
0.5
0.5
0.
5
S2
0.35
0. 0.3
4
5
S1
3
0.
0.3
S0
0.7
0.150.1
0.2
5
Estimated labeling efficiency
S [Observed]
0.3
0.35
0.15
0.8
0.2
TMixt
THeavy
pTPSLPTPPTR
5
0.2
0.9
TLight
Direct
0.2
0. 0. 0.3
6 5 00.4 0
.4 .35
5
pTPSLPTPPTR
0.8
0.9
1
1.1
0.9
1
1.1
1.2
1.13.4
1.5
1.6
0.06
0.08
0.1
0.12
0.14
1.2
1.3
1.4
1.5
1.6
7
1.
0.16
0.18
Error/Signal
Fold change: 2.49 [2.05 3.19]
ASGQAFELILpSPR
KBackground
Direct
Fold change: -1.01 [-1.7 2.5]
TDHGAEIVYKpSPVVpSGDTpSPR
KBac kgr ound
Direct
TLight
TLight
TMixt
TMixt
THeavy
THeavy
S [Observed]
S [Observed]
S0
S1
S2
S3
S4
S5
S6
ASGQAFELILpSPR
S7
S8
KBackground
S0
S1
S3
S4
S5
S6
TMixt
THeavy
S [Observed]
S [Observed]
S2 S3
S4 S5
S6
S7
S8
TLight
TMixt
THeavy
S0 S1
S7
KBac kgr ound
Inverted
TLight
Inverted
S2
TDHGAEIVYKpSPVVpSGDTpSPR
S8
S0
S1
S2
S3
S4
S5
S6
S7
S8
Figure S5 Examples of experimentally observed spectra pairs are shown in relationship to the
contour plot delineating the average fold change error given the Error/Signal-ration and labeling
efficiency estimated by the method presented.
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