Supplementary material to Pedersen et al.: Proteomic characterization of a
temperature sensitive conditional lethal in Drosophila melanogaster.
Titles and legends to figures in supplementary material
Figure S1
Unused ProtScore for all identified proteins in the 5 iTRAQ experiments. An Unused
ProtScore of 1.3 was used as a threshold in all experiments. The Unused ProtScore
estimates the confidence by which a protein can be validated, using only peptide evidence
that is not better explained by a higher ranking protein. An Unused ProtScore of 1.3
corresponds to an identification confidence of 95%, and an Unused ProtScore of 2.0
corresponds to a confidence of 99%. The Unused ProtScore and the identification
confidence are related according to following equation Unused ProtScore = - log (1 percent confidence/100).
Figure S2
Protein sequence coverage (%) for all identified proteins in the 5 iTRAQ experiments.
Figure S3
Expression levels of a subset of proteins identified in experiment 3. Proteins related to
mitochondria are shown in A) and some of the other differentially regulated proteins in
B). Expression level for each protein at the different temperatures is relative to the IC-line
at the permissive temperature. Stars indicate significance level (* = P < 0.05, ** = P <
0.01, *** = P < 0.001) relative to the IC-line at the permissive temperature (20°C). Level
of IC-line at the permissive temperature is indicated by the dashed horizontal line.
2
Figure S1
100
Unused ProtScore
80
60
40
Mean = 9.2
Median = 4.4
20
0
All identified proteins
3
Figure S2
100
% protein coverage
80
60
Mean = 39.36%
Median = 36.27%
40
20
0
All identified proteins
4
0.0
5
*
*
***
B
Adh related
Turandot A
*
1.8
IC-line at 29°C
L-line at 20°C
L-line at 29°C
Turandot C
**
A
Cytochrome c oxidase
mitochondrial precursor
**
*
*
2.0
Putative ATP synthase
f chain, mitochondrial
ATP synthase
subunit d
CG3731
ATP synthase alpha chain
mitochondrial precursor
***
***
***
***
***
*
***
1.4
Stress sensitive B
**
***
1.2
Porin
Relative protein regulation (relative to IC20)
Figure S3
1.6
1.0
0.8
0.6
Table S1
Gene-Enrichment-Analysis of the significantly differentially expressed proteins between
the L-line and the IC-line at permissive temperature (20°C) from experiment 3. The
number of genes identified within specific groups, the percentage of total genes within
groups and the EASE score P-value for the Gene Ontology Themes ‘Biological Process’,
6
EASE score
P-value
Biological Process
No significant biological processes
Molecular Function
Transporter activity
Transmembrane transporter activity
Active transmembrane transporter activity
Cellular Compartment
Mitochondrial part
Mitochondrion
Mitochondrial membrane
Mitochondrial envelope
Lipid particle
Organelle envelope
Envelope
% of group
# of genes
‘Molecular Function’ and ‘Cellular Compartment’.
9
7
5
22.0
17.1
12.2
1.20E-02
2.70E-02
3.70E-02
11
12
6
6
8
6
6
26.8
29.3
14.6
14.6
19.5
14.6
14.6
8.80E-04
4.40E-03
2.70E-02
3.50E-02
3.70E-02
3.90E-02
3.90E-02
Table S2
Gene-Enrichment-Analysis of the significantly differentially expressed proteins between
the L-line and the IC-line at restrictive temperature (29°C) from experiment 3. The
number of genes identified within specific groups, the percentage of total genes within
groups and the EASE score P-value for the Gene Ontology Themes ‘Biological Process’,
7
EASE score
P-value
% of group
Biological Process
Cellular lipid metabolic process
Lipid metabolic process
Monocarboxylic acid metabolic process
Fatty acid metabolic process
Coenzyme metabolic process
Fatty acid beta-oxidation
Fatty acid oxidation
Cofactor metabolic process
Organic acid metabolic process
Carboxylic acid metabolic process
Cellular carbohydrate metabolic process
Molecular Function
Oxireductase activity, acting on the CH-OH group of
donors, NAD or NADP as acceptor
Oxireductase activity
Oxireductase activity, acting on the CH-OH group of
donors
Transferase activity
Cellular Compartment
Intracellular membrane-bound organelle
Membrane-bound organelle
Mitochondrion
# of genes
‘Molecular Function’ and ‘Cellular Compartment’.
10
10
9
6
11
4
4
11
12
12
11
18.5
18.5
16.7
11.1
20.4
7.4
7.4
20.4
22.2
22.2
20.4
9.00E-05
7.10E-04
2.10E-03
1.30E-02
1.30E-02
1.40E-02
1.40E-02
1.80E-02
2.00E-02
2.00E-02
4.60E-02
8
17
14.8
31.5
1.10E-02
2.70E-02
8
12
14.8
22.2
4.10E-02
4.20E-02
18
18
13
33.3
33.3
24.1
4.10E-03
4.10E-03
1.60E-02
Table S3
List of differentially regulated proteins in response to the restrictive temperature in the lethal-line (L-line). P-value from t-test and FlyBase accession
number are listed. ‘Quantification’ column indicates # of peptides used in the t-test, ‘Identification’ column indicates the # of unique but potentially
overlapping peptides identifying the protein. Proteins involved in oxidative phosphorylation (ox) and associated to the mitochondria (m) are indicated
m
ox
m
m
ox
m
m
ox
m
m
m
560
2439
1556
104
93
176
49
362
284
1249
76
278
37
629
539
11
593
237
124
60
87
43
24
22
15
18
25
28
74
17
30
3
44
44
2
70
18
13
Cytological
position
17A9-17A9
35B3-35B3
80A-80B
88E12-88E13
88E13-88E13
91F1-91F1
36B1-36B1
98F12-98F13
9E10-9F1
102D1-102D1
1B4-1B4
85B7-85B7
14B8-14B8
66C8-66C8
85A5-85A5
6C5-6C5
46B4-46B4
32B1-32B1
44B3-44B3
L29/L20
(115/117)
ox
P value
2.15E-28
1.08E-22
8.78E-10
1.91E-09
2.18E-08
3.16E-08
6.61E-08
2.14E-07
3.83E-07
6.97E-07
9.50E-07
5.91E-06
6.57E-06
1.95E-05
2.67E-05
4.40E-05
8.15E-05
1.35E-04
2.56E-04
L29/IC20
(115/116)
M
L20/IC20
(117/116)
Ox
IC29/IC20
(114/116)
Transferrin 1
Adh-related
Sarcoplasmic calcium-binding protein 1
Tropomyosin 1
Tropomyosin 2
ATP synthase, subunit d
Myosin heavy chain
Phosphoglyceromutase
Stress-sensitive B
ATP synthase-β
Cytochrome P450 4g1
CG11963
CG3560
Isocitrate dehydrogenase
CG8036
CG3192
CG1516
Porin
Obp44a
FlyBase ID
FBgn0022355
FBgn0000056
FBgn0020908
FBgn0003721
FBgn0004117
FBgn0016120
FBgn0086783
FBgn0014869
FBgn0003360
FBgn0010217
FBgn0010019
FBgn0037643
FBgn0030733
FBgn0001248
FBgn0037607
FBgn0029888
FBgn0027580
FBgn0004363
FBgn0033268
Identification
Protein name
Quantification
based on the DAVID Gene-Enrichment-Analysis.
1.131
1.040
1.053
0.769
0.748
1.209
1.026
0.934
1.017
1.037
1.127
0.998
1.201
0.974
1.088
1.262
0.921
1.061
0.948
0.826
1.079
0.926
0.895
0.844
1.390
2.030
0.921
1.410
1.142
1.602
0.832
1.655
0.939
0.931
2.232
0.815
1.424
1.141
1.277
1.286
1.044
0.964
0.967
1.345
1.244
1.007
1.303
1.087
1.188
0.951
1.561
0.846
0.944
2.054
0.837
1.365
0.963
1.548
1.200
1.131
1.073
1.136
0.979
0.622
1.098
0.924
0.961
0.736
1.142
0.957
0.901
1.015
0.934
1.031
0.966
0.841
Error
factor
1.042
1.025
1.020
1.095
1.085
1.111
1.139
1.043
1.051
1.027
1.142
1.045
1.107
1.032
1.036
1.198
1.038
1.056
1.062
Myosin light chain alkali
Larval visceral protein H
CG9090
CG4769
Stress-inducible humoral factor Turandot A
Esterase 6 precursor
CG9029
CG9468
CG3321
CG18815
Senescence marker protein-30
CG3731
Accessory gland-specific peptide 26Aa
Probable phosphoserine aminotransferase
CG10512
Troponin C 41F
Ribosomal protein S18
Histone H4 replacement
Malic enzyme
Cytochrome c oxidase polypeptide Va
Ribosomal protein L12
CG16712
CG33138
Stress-inducible humoral factor Turandot C
ATP synthase B chain, mitochondrial precursor
CG8343
FBgn0002772
FBgn0002570
FBgn0034497
FBgn0035600
FBgn0028396
FBgn0000592
FBgn0031746
FBgn0032069
FBgn0038224
FBgn0042138
FBgn0038257
FBgn0038271
FBgn0002855
FBgn0014427
FBgn0037057
FBgn0033027
FBgn0010411
FBgn0013981
FBgn0002719
FBgn0019624
FBgn0034968
FBgn0031561
FBgn0053138
FBgn0044812
FBgn0019644
FBgn0040502
ox
m
m
ox
m
ox
m
ox
m
ox
m
4.11E-04
4.70E-04
5.27E-04
5.31E-04
1.12E-03
1.20E-03
1.38E-03
1.68E-03
2.38E-03
2.73E-03
3.27E-03
3.28E-03
3.66E-03
3.71E-03
3.80E-03
4.79E-03
4.87E-03
5.00E-03
5.14E-03
5.21E-03
5.88E-03
6.70E-03
7.14E-03
7.44E-03
7.63E-03
7.66E-03
9
18
17
61
41
70
78
75
25
20
27
79
81
71
8
19
58
25
6
308
90
10
36
151
45
31
19
3
9
10
8
10
15
19
9
2
6
12
10
14
5
4
8
7
2
30
10
4
4
28
7
7
2
98A14-98A15
44D1-44D1
56F15-56F16
64C13-64C13
93A2-93A2
69A1-69A1
26A1-26A1
29F1-29F1
88B4-88B4
68D1-68D1
88D2-88D2
88D6-88D6
26A1-26A1
99A1-99A1
78C2-78C2
41F2-41F3
56F11-56F11
88C9-88C9
87C6-87C7
86F9-86F9
60B7-60B7
24B3-24B3
49F4-49F4
93A2-93A2
67C5-67C5
42A13-42A13
1.284
1.044
1.092
1.253
1.304
1.099
1.159
0.630
1.114
0.841
1.179
1.158
0.879
0.772
1.016
1.002
0.923
0.385
0.988
1.147
0.984
0.719
0.886
1.971
1.083
1.091
1.221
1.257
1.484
1.932
0.482
1.387
0.869
1.911
1.396
0.885
1.186
1.501
0.962
0.486
0.845
1.157
1.200
0.471
0.875
1.463
1.291
1.014
1.158
0.383
1.622
0.795
0.909
0.810
1.304
1.779
0.455
2.021
1.407
0.678
1.396
1.068
1.131
1.439
1.063
0.537
1.054
0.900
0.930
0.471
0.986
1.442
0.931
0.942
0.933
0.499
1.396
0.486
0.759
0.655
0.881
0.930
0.949
1.473
1.602
0.361
1.005
1.205
0.962
0.964
1.105
1.102
1.254
0.786
0.779
1.007
1.128
0.996
0.733
0.939
0.811
1.298
0.873
0.621
1.209
1.191
1.103
1.154
1.161
1.115
1.208
1.311
1.072
1.182
1.111
1.110
1.107
1.244
1.136
1.142
1.159
1.391
1.049
1.097
1.331
1.132
1.075
1.255
1.172
1.188
Table S4
Proteins significantly differentially expressed between the L-line and the IC-line at the permissive temperature (20°C)
Protein name
Larval serum protein 2
Glutamate oxaloacetate transaminase 2
MET75Cb protein precursor
ATP synthase alpha chain, mitochondrial precursor
Voltage-dependent anion-selective channel
CG31883
CG31305
CG6287
Stress sensitive b
Heat shock protein 60
Probable acyl-CoA dehydrogenase, medium-chain specific, mitochondrial precursor
CG8626
CG18067
Neural conserved at 73EF
Serine protease inhibitor
Ejaculatory bulb specific protein III precursor
CG15369
CG30415
CG8343
CG9914
Aldehyde dehydrogenase
CG10924
Thiolester containing protein IV
Succinyl-CoA ligase alpha-chain, mitochondrial precursor
CG10664
CG11151
Cytochrome P450 4g1
FlyBase ID
FBgn0002565
FBgn0001125
FBgn0028415
FBgn0011211
FBgn0004363
FBgn0032122
FBgn0037913
FBgn0032350
FBgn0003360
FBgn0015245
FBgn0035811
FBgn0034152
FBgn0034512
FBgn0010352
FBgn0028986
FBgn0011695
FBgn0030105
FBgn0250838
FBgn0040502
FBgn0030737
FBgn0012036
FBgn0034356
FBgn0041180
FBgn0004888
FBgn0032833
FBgn0030519
FBgn0010019
10
L20/IC20
(117/116)
0.73
0.83
1.14
1.09
1.16
0.49
1.13
0.85
1.17
0.87
0.86
0.81
0.83
0.91
1.17
1.12
1.22
1.37
0.90
1.14
0.94
0.83
1.27
0.92
1.12
1.12
1.14
P-value
5.00E-16
1.24E-05
1.81E-05
2.35E-04
3.49E-04
4.00E-04
4.22E-04
5.31E-04
7.45E-04
8.65E-04
9.34E-04
1.20E-03
2.01E-03
2.65E-03
3.20E-03
3.27E-03
3.30E-03
4.76E-03
6.24E-03
6.45E-03
8.52E-03
1.01E-02
1.08E-02
1.34E-02
1.40E-02
1.66E-02
1.87E-02
Error
factor
1.06
1.09
1.06
1.04
1.08
1.45
1.07
1.08
1.09
1.08
1.09
1.12
1.06
1.06
1.10
1.07
1.09
1.20
1.06
1.09
1.05
1.15
1.19
1.07
1.06
1.10
1.11
CG6188
ATP synthase , subunit d
CG11255
Sorbitol dehydrogenase
Dihydropteridine reductase
Adh-related
Fructose-bisphosphate aldolase
CG7966
CG12321
Phosphatidylinositol transfer protein
CG12082
CG15616
40S ribosomal protein S20
Guanine nucleotide-binding protein gamma-e subunit
FBgn0038074
FBgn0016120
FBgn0036337
FBgn0022359
FBgn0035964
FBgn0000056
FBgn0000064
FBgn0038115
FBgn0038577
FBgn0026158
FBgn0035402
FBgn0034153
FBgn0019936
FBgn0028433
1.30
1.18
0.80
0.84
1.18
1.39
1.03
1.34
1.13
0.81
0.98
0.86
0.82
1.45
1.87E-02
1.97E-02
2.01E-02
2.07E-02
2.22E-02
2.33E-02
2.69E-02
2.82E-02
3.00E-02
3.29E-02
3.37E-02
3.64E-02
3.75E-02
4.90E-02
1.10
1.15
1.19
1.14
1.15
1.31
1.03
1.27
1.07
1.15
1.01
1.15
1.19
1.44
Table S5
Proteins significantly differentially expressed between the L-line and the IC-line at the restrictive temperature (29°C)
Protein name
MET75Cb protein precursor
Glutamate oxaloacetate transaminase 2
Sarcoplasmic calcium-binding protein 1
Arginine kinase
CG31883
Larval serum protein 2
Adh-related
Ductus ejaculatorius peptide 99B precursor
Voltage-dependent anion-selective channel
Isocitrate dehydrogenase
Odorant-binding protein 99b
FlyBase ID
FBgn0028415
trm|Q8IPY3
FBgn0020908
FBgn0000116
FBgn0032122
FBgn0002565
FBgn0000056
FBgn0250832
FBgn0004363
FBgn0001248
FBgn0039685
11
L29/IC29
1.27
0.82
0.93
0.94
0.44
0.84
1.66
1.25
1.16
0.92
1.35
P-value
8.31E-16
8.55E-12
1.19E-08
1.13E-06
1.41E-06
2.42E-05
5.37E-05
7.12E-05
7.52E-05
1.27E-04
3.31E-04
Error
factor
1.05
1.05
1.03
1.03
1.35
1.08
1.20
1.10
1.07
1.04
1.16
CG8036
Mitochondrial aconitase
CG15616
Esterase 6 precursor
CG11151
Aldehyde dehydrogenase
Accessory gland-specific peptide 70A precursor
Glutamine synthetase 1
CG30415
Acyl-CoA-binding protein homolog
CG3731
CG9914
Heat shock protein 60
CG15369
CG7920
Glycogen phosphorylase
6-phosphogluconate dehydrogenase, decarboxylating
Protein anoxia up-regulated
Glutathione S-transferase S1
CG8343
CG5844
CG6287
CG32920
CG9391
Putative ATP synthase f chain, mitochondrial
60S acidic ribosomal protein P0
Heterogeneous nuclear ribonucleoprotein at 87F
6-phosphofructo-2-kinase
Glycerol-3-phosphate dehydrogenase [NAD+], cytoplasmic
Probable acyl-CoA dehydrogenase, mitochondrial precursor
CG6045
Yippee interacting protein 2
Cytochrome c-2
FBgn0037607
FBgn0010100
FBgn0034153
FBgn0000592
FBgn0030519
FBgn0012036
FBgn0003034
FBgn0001142
FBgn0250838
FBgn0010387
FBgn0038271
FBgn0030737
FBgn0015245
FBgn0030105
FBgn0039737
FBgn0004507
FBgn0004654
FBgn0020439
FBgn0010226
FBgn0040502
FBgn0038049
FBgn0032350
FBgn0038570
FBgn0037063
FBgn0035032
FBgn0000100
FBgn0004237
FBgn0027621
FBgn0001128
FBgn0035811
FBgn0038349
FBgn0040064
FBgn0000409
12
0.93
0.94
0.87
1.29
1.11
0.93
0.89
0.84
1.21
0.86
1.11
1.11
0.89
1.22
0.93
0.96
0.89
1.56
0.94
0.70
0.69
0.88
0.92
1.17
1.18
1.22
1.27
0.89
0.95
0.90
0.91
0.93
1.09
4.86E-04
7.38E-04
9.10E-04
1.24E-03
4.53E-03
4.88E-03
6.78E-03
7.40E-03
7.72E-03
8.10E-03
8.31E-03
8.45E-03
8.88E-03
9.26E-03
9.47E-03
1.22E-02
1.24E-02
1.35E-02
1.46E-02
1.46E-02
1.53E-02
1.60E-02
1.67E-02
1.77E-02
1.91E-02
2.02E-02
2.04E-02
2.28E-02
2.50E-02
2.51E-02
2.51E-02
2.91E-02
3.12E-02
1.04
1.03
1.08
1.16
1.07
1.05
1.08
1.13
1.14
1.12
1.08
1.08
1.09
1.13
1.05
1.03
1.10
1.25
1.05
1.29
1.12
1.11
1.07
1.06
1.14
1.18
1.10
1.08
1.05
1.09
1.09
1.07
1.08
CG5162
CG10512
CG17838
CG4347
Fructose-bisphosphate aldolase
CG4389
Glutathione S transferase D1
CG6439
Triose phosphate isomerase
FBgn0030828
FBgn0037057
FBgn0038826
FBgn0035978
FBgn0000064
FBgn0028479
FBgn0001149
FBgn0038922
FBgn0086355
13
1.16
1.11
1.14
1.13
0.97
0.92
0.89
1.08
0.95
3.24E-02
3.24E-02
3.25E-02
3.44E-02
3.73E-02
4.44E-02
4.56E-02
4.74E-02
4.75E-02
1.14
1.10
1.12
1.12
1.03
1.09
1.12
1.08
1.05