Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2deoxyglucose using pulsed stable isotope-resolved metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Figure S 1 - Experimental setup of harvesting procedure for a pSIRM cell culture experiment.
Cells are seeded with pre-determined cell number to avoid growth inhibitory effects until harvest on
day 3 of the experiment. Media is replaced to supply cells with nutrients 24 and 4 hours prior the
harvest. The harvest protocol summarizes the labeling procedure of adherent cell cultures (see
Methods section).
1
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2deoxyglucose using pulsed stable isotope-resolved metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Table S 1 – Composition of the full label medium and label buffer for pSIRM experiments used for
the labeling of T98G cells and other cell lines as mentioned in the manuscript.
Base
Carbon sources
Supplemented with
Full label medium
Dulbeccos modified eagle medium (DMEM)
- without Glucose
- without Glutamine
- without Pyruvate
+ 13C-Glucose (2.5 g/L)
+ 12C-Glutamine (4 mM)
+ small molecules (inhibitor, antibiotics etc.)
Label buffer
HEPES (5 mM)
NaCl (140 mM)
pH 7.4
+ 13C-Glucose (2.5 g/L)
+ 12C-Glutamine (4 mM)
2
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2deoxyglucose using pulsed stable isotope-resolved metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Figure S 2 – Experimental scheme of the reproducibility experiment to determine technical and
biological variances of GC-MS derived data.
T98G cells were cultured for three days, and labeled with 13C-glucose for 3 minutes. Cell extracts were
prepared separately for each cell culture plate. Technical variance was determined by the repeated
measurement of pooled cell samples. Biological reproducibility was determined by repeated
measurement of five independently treated cell samples.
3
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2deoxyglucose using pulsed stable isotope-resolved metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Table S 2 – Biological and technical variation of the measurement of metabolite pool sizes in T98G
cells.
The coefficient of variation (CoV) of the peak area for each metabolite was calculated from six
consecutive measured technical replicates. The calculation of CoVs for the biological reproducibility
based on five biological replicates, each measured in four technical repetitions.
Compound
Adenine
Alanine, betaAlanine
Asparagine
Aspartic acid
Butanoic acid, 2-aminoButanoic acid, 3-hydroxyButanoic acid, 4-aminoCitric acid
Cytosine
Fructose-1,6-bisphosphate
Fructose
Fructose-6-phosphate
Fumaric acid
Glucose
Glucose-6-phosphate
Glutamine
Glutaric acid, 2-hydroxyGlutaric acid, 2-oxoGlyceric acid
Glyceric acid-3-phosphate
Glycerol
Glycerol-3-phosphate
Glycine
Inositol, myoIsoleucine
Lactic acid
Leucine
Lysine
Malic acid
Methionine
Pantothenic acid
Phosphoenolpyruvic acid
Proline
Putrescine
Pyroglutamic acid
Pyruvic acid
Ribitol
Ribose-5-phosphate
Serine
Succinic acid
Sucrose
Threonine
Tyrosine
Uracil
Uridine 5'-monophosphate
Uridine
Valine
Coefficient of variation
Technical
Biological
replicates replicates
13.7
14.7
8.9
12.4
6.8
10.0
9.0
5.3
3.6
10.3
9.3
5.5
7.6
15.9
16.1
19.6
8.9
10.6
15.1
12.1
15.3
15.6
11.5
13.9
6.4
10.3
9.0
10.4
8.6
7.0
10.8
11.6
5.9
11.9
8.7
13.4
12.2
14.3
6.3
7.1
11.3
14.7
15.3
16.5
10.2
13.1
12.7
6.6
11.3
11.6
8.1
12.0
3.9
11.0
7.6
8.6
12.0
11.5
9.2
13.3
16.8
20.7
11.2
11.2
11.8
13.2
7.6
5.2
14.5
8.1
10.1
13.8
5.3
7.5
11.5
9.3
11.3
11.2
10.0
10.2
15.0
21.3
23.2
29.5
10.4
11.1
10.7
8.2
10.0
20.5
6.8
14.3
21.5
13.5
5.6
11.6
4
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2deoxyglucose using pulsed stable isotope-resolved metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Table S 3 – Quant mixture composition and concentration range for each metabolite
Compound
Adenine
Adenosine
Alanine
Alanine, beta
Arginine
Asparagine
Aspartic acid
Butyric acid, 3-hydroxy
Butyric acid, 4-amino
Citric acid
Creatinine
Cysteine
Cytosine
Dihydroxyacetone phosphate
Erythritol, meso
Fructose
Fructose-1,6-bisphosphate
Fructose-6-phosphate
Fumaric acid
Gluconic acid-6-phosphate
Glucosamine
Glucose
Glucose 1-phosphate
Glucose-6-phosphate
Glutamic acid
Glutamine
Glutaric acid
Glutaric acid, 2-hydroxy
Glutaric acid, 2-oxo
Glyceraldehyde-3-phosphate
Glyceric acid
Glyceric acid-3-phosphate
Glycerol
Glycerol-3-phosphate
Glycine
GMP
Hypotaurine
Inosine
Inositol, myo
Isoleucine
Lactic Acid
Leucine
Lysine
Malic acid
Methionine
Pantothenic acid
Phenylalanine
Phosphoenolpyruvic acid
Proline
Putrescine
Pyroglutamic acid
Concentration range in Quant mix (pmol)
Minimum
Maximum
37
7400
94
18709
673
134695
56
11225
57
11481
114
22707
75
15025
144
28818
48
9697
260
52051
221
44201
41
8254
45
9001
441
88183
409
81887
416
83259
271
54288
66
13154
172
34462
73
14616
23
4638
1943
388544
59
11894
164
32884
680
135934
684
136855
151
30278
286
57268
171
34223
323
64668
80
15987
217
43478
326
65154
135
26996
333
66605
101
20113
92
18323
56
11184
278
55506
191
38116
2231
446190
457
91484
103
20531
224
44746
34
6702
84
16788
242
48429
75
15036
304
60801
31
6208
465
92937
5
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2deoxyglucose using pulsed stable isotope-resolved metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Pyruvate
Ribose
Ribose, 2-deoxy
Ribose 5-phosphate
Serine
Succinic acid
Threonine
Tryptophan
Tyrosine
Uracil
Uridine 5′-monophosphate
Valine
1454
100
37
456
571
212
839
49
55
134
407
213
290803
19983
7455
91208
114188
42341
167898
9793
11038
26764
81489
42680
6
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2deoxyglucose using pulsed stable isotope-resolved metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Figure S 3 - Illustration of quant-addition experiment.
The measurement of the quant mixture delivers calibration curves, which were used to quantify the
biological sample – here shown for citric acid and glycerol-3-phosphate. The quantification results are
shown for the individual measurements of quantification mixtures (black), cell samples (green), the
sum of quantities of cell extracts and quantification mixtures (red), and the measurements of samples
spiked into quantification mixtures (blue).
7
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2-deoxyglucose using pulsed stable isotope-resolved metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Table S 4 – Data quant addition experiment. T98G cell extracts, equivalent to 7.14 x 105 cells, were quantified by external quantification. Same amounts of extracts
were spiked into each dilution of the quantification mixture.
Compound
Adenine
Alanine, betaAlanine
Butanoic acid, 3-hydroxyCitric acid
Cytosine
Erythritol
Fructose BP
Fructose MP
Fructose-6-phosphate
Fumaric acid
Glucose-1/6-phosphate
Glutaric acid, 2-hydroxyGlutaric acid, 2-oxoGlutaric acid
Glyceric acid
Glyceric acid-3-phosphate
Glycerol
Glycerol-3-phosphate
Glycine
Hypotaurine
Inositol, myoLactic acid
Leucine
Malic acid
Pantothenic acid
Phenylalanine
Proline
Putrescine
Pyroglutamic acid
Pyruvic acid
Ribose
Serine
Succinic acid
Threonine
Uracil
Valine
Concentration in
extract in pmol
(Avg ± SD)
614
± 182
2300 ± 624
9840 ± 3183
213
±
88
4333 ± 330
1467 ± 326
1403 ± 147
9224 ± 473
8791 ± 793
326
±
37
1234 ±
93
1137 ± 207
396
±
25
6150 ± 380
136
±
51
223
±
24
875
± 139
4305 ± 369
1111 ± 142
12759 ± 3200
2551 ± 273
7313 ± 885
189454 ± 18268
16789 ± 3287
5428 ± 321
1396 ± 244
10229 ± 2156
7661 ± 1824
151
±
12
58365 ± 4119
31775 ± 5243
91
±
27
23025 ± 3328
1801 ± 134
36780 ± 5628
4135 ± 2397
11337 ± 2111
Conc. quant (measured) + conc. extract (pmol)
1:200
614
2300
10689
370
4609
1467
1869
9678
9225
457
1431
1494
680
6150
265
299
875
4568
1315
13085
2665
7655
192498
16789
5676
1488
10632
7661
198
58818
32523
183
23674
2011
37576
4135
11559
1:100
614
2434
11537
529
4855
1753
2266
10060
9615
533
1617
1613
960
6529
443
377
1436
4981
1406
13507
2770
7878
194432
17776
5884
1569
10823
7962
238
59351
35292
289
24376
2260
38779
4403
11847
1:50
807
2560
12291
780
5225
1857
2987
10707
10287
613
1929
1915
1531
6787
733
521
1628
5732
1594
14327
2913
8301
197362
18899
6262
1715
11110
8766
311
60187
38762
502
25174
2679
40274
4722
12214
1:20
950
2883
16541
1717
6596
1994
5228
12880
12481
912
2848
2881
3345
7852
1618
1001
2795
7979
2228
16497
3438
9637
208360
22799
7462
2175
12534
12088
499
62939
48600
1086
28578
4098
45846
5428
13616
1:10
1371
3456
24554
3263
9141
2347
9093
17166
16838
1501
4585
4954
6495
9624
3248
1792
4864
11706
3482
20261
4502
12310
229315
28801
9561
2990
15479
17110
823
67892
66948
2114
35000
6439
56066
6721
15970
1:5
2184
4497
36271
5977
15188
3224
17585
26803
26351
2980
8242
10100
12605
13348
6360
3463
9482
17908
6402
26574
6506
18742
280861
35738
14172
4917
20126
20495
1412
77985
95473
4566
45122
10545
71285
9606
19668
Conc. (Quant + spiked in extract) (pmol)
1:2
4220
8130
83215
16149
29647
5995
43782
49782
50400
7169
18609
24578
29013
22607
15488
8443
22350
37709
14642
46330
11552
35335
432389
63458
27697
9710
35598
39253
3415
105173
178251
9154
82883
23352
125978
17499
35039
1:200
697
2078
11637
458
4635
1492
1813
9809
9334
311
1462
1332
783
6284
317
309
1090
4785
1236
12253
2617
7811
194868
18069
5682
1597
10559
8525
186
56276
35120
230
24334
2040
38110
2044
11936
1:100
576
1987
8756
507
4992
1585
2390
10729
10040
363
1744
1489
1277
6766
561
439
1196
5242
1389
10957
2751
8279
195966
16244
6436
1740
10023
7189
221
61889
35723
347
24116
2373
37739
1258
11267
1:50
791
2275
11316
1000
6119
1975
3651
12542
11675
436
2357
1837
2410
7550
1002
696
1697
6415
1839
12341
3241
9033
218580
20882
7568
2090
11674
10088
325
68508
44233
656
28823
3233
45000
900
14342
1:20
1128
2722
15642
2238
7847
2649
6710
15474
14497
723
3621
3155
5245
8952
2319
1398
2860
9767
2729
15094
4034
11657
246012
25246
9418
3099
13275
13832
552
76717
64988
1313
35298
5268
55794
1887
16885
1:10
1466
3115
22001
4209
10398
3233
11466
19566
18623
1118
5852
5141
9134
10693
4442
2476
4455
13930
4025
17442
5103
14604
269882
30916
12320
4038
15093
18334
872
83975
98394
2422
42207
8186
67967
4457
19559
1:5
2032
4105
34702
7850
14948
4266
21274
27418
26486
2030
9728
8372
15837
13739
8281
4456
7944
22057
6736
22535
6938
21199
310055
37820
17703
6077
17457
24202
1552
97376
141628
4185
53976
13249
91359
6908
23046
1:2
4422
6419
83667
19883
30685
7492
49021
50961
51298
4996
20213
19299
33714
22961
18164
9674
19644
45069
14917
36278
12927
39071
424055
63263
32113
11764
34055
43926
3353
125674
259046
8674
93783
26145
158356
14402
33794
8
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2deoxyglucose using pulsed stable isotope-resolved metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Table S 5 - Recovery of metabolites
The recovery is defined as ratio of the quantity of spike-in and summed up quantities of
individual measured quantification mixtures and extracts. The recovery was calculated at equal
concentrations of quantification mixture and extracts.
Compound
Adenine
Alanine, betaAlanine
Butanoic acid, 3-hydroxyCinnamic acid, transCitric acid
Cytosine
Erythritol
Fructose BP
Fructose MP
Fructose-6-phosphate
Fumaric acid
Glucose-1/6-phosphate
Glutaric acid, 2-hydroxyGlutaric acid, 2-oxoGlutaric acid
Glyceric acid
Glyceric acid-3-phosphate
Glycerol
Glycerol-3-phosphate
Glycine
Hypotaurine
Inositol, myoLactic acid
Leucine
Malic acid
Pantothenic acid
Phenylalanine
Proline
Putrescine
Pyroglutamic acid
Pyruvic acid
Ribose
Serine
Succinic acid
Threonine
Uracil
Valine
Recovery
(%)
112.9
91.3
92.1
109.8
104.7
116.4
132.3
122.2
108.1
110.6
75.3
102.2
102.7
124.1
107.0
119.6
125.0
104.2
111.9
122.5
84.8
113.4
115.9
104.2
106.6
126.9
135.1
86.7
110.8
104.5
119.5
148.3
125.6
119.6
124.6
128.2
69.1
106.8
9
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2deoxyglucose using pulsed stable isotope-resolved metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Table S 6 – Intracellular metabolite concentrations of T98G cells.
Data is derived from the reproducibility experiment (see Methods section). T98G cells were
incubated with 13C6-glucose for three minutes.
Compound
Adenine
Alanine
Alanine, betaAsparagine
Aspartic acid
Butanoic acid, 2-amino
Butanoic acid, 3-hydroxy
Citric acid
Fructose
Fructose-1,6,diphosphate
Fructose-6-phosphate
Fumaric acid
Glucose-6-phosphate
Glutaric acid, 2-hydroxy
Glutaric acid, 2-oxo
Glyceric acid
Glyceric-acid-3-phosphate
Glycerol-3-phosphate
Glycine
Inositol, myo
Isoleucine
Lactic acid
Leucine
Lysine
Malic acid
Methionine
Pantothenic acid
Proline
Putrescine
Pyruvic acid
Ribose-5-phosphate
Serine
Succinic acid
Threonine
Tyrosine
Uracil
Uridine-5-monophosphate
Valine
Quantity
(pmol / 106 cells)
Average ± STDEV
77
±
15
841
±
89
623
±
122
202
±
15
251
±
22
3,965
±
702
51
±
7
327
±
17
1,068
±
32
773
±
45
48
±
5
135
±
6
532
±
27
98
±
5
235
±
6
30
±
2
133
±
7
90
±
5
2,332
±
135
1,203
±
20
1,960
±
170
9,873
±
1,269
2,409
±
134
1,512
±
238
547
±
22
1,009
±
74
142
±
3
892
±
56
36
±
1
195
±
31
185
±
11
2,546
±
154
30
±
8
4,971
±
339
1,101
±
173
111
±
21
8,410
±
615
333
±
17
13
C-Quantity
(pmol / 106 cells)
Average ± STDEV
n.d
±
n.d
10.3
±
3.7
n.d
±
n.d
n.d
±
n.d
0.4
±
0.5
n.d
±
n.d
n.d
±
n.d
3.7
±
2
29.0
±
2.6
n.d
±
n.d
10.8
±
0.6
0.4
±
0.2
100.6
±
6.3
0.5
±
0.3
2.3
±
1.5
n.d
±
n.d
n.d
±
n.d
1.6
±
0.7
4.0
±
2.4
n.d
±
n.d
n.d
±
n.d
454.5
±
69.9
n.d
±
n.d
n.d
±
n.d
1.2
±
1.1
n.d
±
n.d
n.d
±
n.d
n.d
±
n.d
n.d
±
n.d
9.3
±
1.5
n.d
±
n.d
n.d
±
n.d
0.1
±
0.1
n.d
±
n.d
n.d
±
n.d
n.d
±
n.d
n.d
±
n.d
n.d
±
n.d
10
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2deoxyglucose using pulsed stable isotope-resolved metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Table S 7 – Metabolite specific mass fragments for the calculation of 13C-isotope incorporation.
Mass fragments are chosen concerning their uniqueness for the derivate of the
metabolite and were evaluated in separately performed 13C6-glucose and 13C5glutamine labeling experiments.
Mass fragment (m/z)
Labeling with
Compound
Derivate
Abbr.
Unlabeled u-13C-glucose
Alanine
3TMS
Ala
188
190
Aspartic acid
3TMS
Asp
232
235 (a)
Citric acid
4TMS
Cit
273
275
Dihydroxyacetone phosphate
1MeOX 3TMS DHAP
400
403
Fructose
1MeOX 5TMS Fru
217
220
Fructose-1,6-bisphosphate
1MeOX 7TMS F16BP
217
220
Fructose-6-phosphate
1MeOX 6TMS F6P
217
220
Fumaric acid
2TMS
Fum
245
247
Glucose
1MeOX 5TMS Glc
319
323
Glucose-6-phosphate
1MeOX 6TMS G6P
217
220
Gluconic acid-6-phosphate
7TMS
PG6
217
220
Glutamic acid
3TMS
Glu
246
Glutamine
3TMS
Gln
156
Glutaric acid
2TMS
Glut
261
Glutaric acid, 2-hydroxy
3TMS
Glut-OH
247
Glutaric acid, 2-oxo
1MeOX 2TMS aKG
198
200
Glyceric acid-3-phosphate
4TMS
3PGA
357
359
Glycerol
3TMS
Glyc
218
221
Glycerol-3-phosphate
4TMS
Glyc3P
357
359
Glycine
3TMS
Gly
276
277
Lactic acid
2TMS
Lac
219
222
Malic acid
3TMS
Mal
233
235
Phosphoenolpyruvic acid
3TMS
PEP
369
372
Pyruvic acid
1MeOX 1TMS Pyr
174
177
Ribose-5-P
1MeOX 5TMS R5P
217
220
Serine
3TMS
Ser
204
206
Succinic acid
2TMS
Suc
247
249
(a) – when simultaneously applied with 13C-glutamine labeling, the resulting 13Cpyroglutamate might interfere with this mass range.
Labeling
with u-13Cglutamine
277
249
250
160
266
251
203
236
251
11
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2deoxyglucose using pulsed stable isotope-resolved metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Figure S 4 - Concentration dependency of mass isotopomer fractions.
The mass isotopomer fraction (MIF) of mass fragment m+0 for citric acid (m/z = 273-279) is
shown for a broad intensity range (black circles). The expected value (red line) was calculated
by Mass Spec Calculator Pro Demo version 4.09 (ChemSW, Inc.). The proportion of m+0 in the
mass range decreases and yields higher precision with increasing levels of citric acid.
12
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2deoxyglucose using pulsed stable isotope-resolved metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Table S 8 – Illustration of the position dependent strategy for correction of natural 13C-isotope
abundance.
13
C1-Glucose and 12C-glucose were mixed in known ratios to proof the position dependent
(targeted) calculation strategy. Two examples are shown for the determination of 13Cincorporation as described.
Intensities in (-)
12C
m/z
10 % 13C1
MIF in (%)
10 %
13C
1
50 %
13C
1
12C
10 %
13C
1
50 %
13C
1
Snat+1 Sinc+1
50 % 13C1
L
Snat+1
Sinc+1
L
9.6
10.2
33.9
50.5
319
m0
156460
153425
84507
66.6
60.2
33.2
320
m+1
48250
63669
112421
20.6
25.0
44.2
18.6
6.4
321
m+2
23579
28289
38071
10.0
11.1
15.0
9.1
2.0
5.0
10.0
322
m+3
5038
7389
15467
2.1
2.9
6.1
1.9
1.0
1.1
5.0
323
m+4
1199
1716
3268
0.5
0.7
1.3
0.5
0.2
0.3
1.0
324
m+5
228
262
751
0.1
0.1
0.3
0.1
0.0
0.0
0.2
Sum
234754
254750
254485
100
100
100
13
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2deoxyglucose using pulsed stable isotope-resolved metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Table S 9 – Validation of correction strategies for the natural 13C-carbon abundance.
1-13C1-Glucose (purity 99%) and unlabeled glucose were mixed in known ratios and measured
in three independent replicates. Uncorrected values overestimate incorporation especially in
the lower range of isotope incorporation, whereas both calculation strategies consider the
natural contribution of carbon-13 correctly.
13
C-abundance in glucose (%)
Expected isotope
abundance (%)
Uncorrected
0
1.98
23.4 ± 0.09
24.7 ± 0.25
4.95
26.4 ± 0.21
9.90
24.75
49.50
29.6 ± 0.20
39.2 ± 0.79
56.8 ± 0.89
10.3
25.3
50.3
±
±
±
74.25
89.10
94.05
76.1 ± 0.78
89.0 ± 0.04
93.9 ± 0.14
74.2
88.6
93.8
97.02
96.8 ± 0.19
99.00
98.7 ± 0.04
Targeted
0.0 ±
2.1 ±
0.16
0.42
5.0 ±
Positionindependent
0.0
2.0
±
±
0.12
0.32
0.36
5.0
±
0.27
0.33
1.19
1.18
10.1
25.0
49.8
±
±
±
0.29
1.26
1.28
±
±
±
0.91
0.04
0.15
74.0
88.4
93.7
±
±
±
0.94
0.03
0.15
96.7
±
0.19
96.7
±
0.20
98.7
±
0.04
98.7
±
0.05
14
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2deoxyglucose using pulsed stable isotope-resolved metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Table S 10 – Biological and technical variation of 13C-glucose incorporation.
T98G cells were incubated with u-13C glucose for three minutes and harvested as described in
the Methods section.
Compound
3PGA
Ala
Cit
F16BP
Fru
F6P
Fum
G6P
Glut-OH
Glyc3P
Gly
Lac
Mal
Pyr
Suc
Mass
357
188
273
217
217
217
245
217
198
357
276
219
245
174
247
Average (13C-Glc incorporation in %)
± STDEV
Technical repl.
Biological repl.
77.7 ± 4.0
83.2 ± 2.1
4.7 ± 1.3
5.5 ± 0.6
4.2 ± 2.0
7.1 ± 1.2
78.8 ± 1.0
79.7 ± 0.7
10.7 ± 0.7
7.7 ± 0.7
84.0 ± 4.8
83.2 ± 4.0
0.9 ± 0.7
0.6 ± 0.2
74.6 ± 0.5
74.3 ± 0.4
3.2 ± 2.4
1.5 ± 1.6
6.7 ± 2.7
8.9 ± 1.3
0.2 ± 0.6
n.d. ± n.d.
17.9 ± 0.4
18.4 ± 2.7
0.7 ± 0.7
0.2 ± 1.1
18.9 ± 3.0
17.3 ± 1.3
n.d. ± n.d.
1.0 ± 1.2
15
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2deoxyglucose using pulsed stable isotope-resolved metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Figure S 5 - Absolute 13C-quantities of intracellular metabolites in T98G cells.
Data derived from the reproducibility experiment (see Methods section and supplement
table 6).
16
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2deoxyglucose using pulsed stable isotope-resolved metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Table S 11 – Comparison of carbon routing within the CCM of different cell lines.
The labeled quantities of metabolites after 13C-Glucose incubation into T98G, HEK293, HeLa and
HCT-116 cells reveal differences in carbon routing in the central carbon metabolism. Averages
and standard deviations for labeled quantities are shown relative to T98G cells.
13
Compound
3PGA
Ala
Cit
DHAP
F1P
F6P
Fru
G6P
Glyc
Glyc3P
Lac
Pyr
R5P
Ser
T98G
1.00 ±
1.00 ±
1.00 ±
1.00 ±
1.00 ±
1.00 ±
1.00 ±
1.00 ±
1.00 ±
1.00 ±
1.00 ±
1.00 ±
1.00 ±
1.00 ±
0.04
0.22
0.20
0.08
0.15
0.06
0.04
0.07
0.03
0.09
0.04
0.02
0.12
0.21
C-Glucose labeled quantity
Average ± STDEV
HEK293
HeLa
2.77 ± 0.08
1.98 ± 0.01
9.59 ± 2.65
2.53 ± 0.65
13.32 ± 0.32
9.82 ± 0.34
2.51 ± 0.26
1.68 ± 0.30
0.84 ± 0.06
0.95 ± 0.21
1.11 ± 0.02
0.73 ± 0.05
0.32 ± 0.01
9.56 ± 0.04
3.75 ± 0.02
2.34 ± 0.04
0.67 ± 0.12
3.96 ± 0.09
15.57 ± 0.86
1.85 ± 0.40
8.88 ± 2.56
6.64 ± 0.02
8.34 ± 1.10
4.18 ± 0.80
1.21 ± 0.13
1.21 ± 0.07
17.29 ± 1.68
5.03 ± 0.61
HCT-116
2.47 ± 0.01
1.62 ± 0.63
17.66 ± 0.35
3.04 ± 0.10
65.64 ± 0.07
10.89 ± 0.05
12.40 ± 0.58
6.82 ± 0.06
4.40 ± 0.58
3.78 ± 0.00
12.78 ± 0.53
10.65 ± 0.24
1.52 ± 0.01
7.06 ± 0.93
17
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2-deoxyglucose using pulsed stable isotope-resolved
metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Table S 12 – 3-Bromopyruvate treatment rearranges central carbon metabolism in T98G cells.
T98G cells were treated for 12 min with 2 mM 3-Bromopyruvate and labeled for 3 minutes with u-13C-glucose. Fold changes of peak intensity and 13Clabel incorporation are shown relative to control. Two-sided, heteroscedastic t-test indicates significant changes (+++ < 0.005, ++ < 0.01, + < 0.05, n.s.
= not significant, n.d. = not detected).
15min
30 min
15min
30 min
15min
30 min
15min
30 min
15min
30 min
15min
30 min
15min
30 min
Label inc. x
intensity
30 min
Peak intensity
T-Test
Label
incorporation
15min
% Change relative to control
Label
Label inc. x
incorporation
intensity
30 min
Peak
intensity
15min
Compound
Fold changes relative to control
Peak
Label
Label inc. x
intensity
incorporation
intensity
G6P
F6P
F1,6-BP
0,8
3,2
3,9
1,7
8,2
3,4
0,34
0,10
0,37
0,25
0,07
0,44
0,28
0,31
1,07
0,43
0,55
1,68
-17
224
285
68
722
240
-66
-90
-63
-75
-93
-56
-72
-69
6,8
-57
-45
68,0
n.s.
+++
++
+++
+++
+
+++
+++
++
+++
+++
+++
+++
+++
n.s.
+++
+++
+
F1P
Fru
6PG
0,7
1,0
4,5
1,2
1,1
5,1
1,13
0,30
n.d.
0,70
0,30
n.d.
0,57
0,30
n.d.
0,59
0,34
n.d.
-26
1
355
21
11
413
13
-70
n.d.
-30
-70
n.d.
-43
-70
n.d.
-41
-66
n.d.
+
n.s.
+++
n.s.
n.s.
+++
n.s.
+++
+
+++
n.s.
+++
n.s.
+++
3PGA
Pyr
Lac
n.d.
1,0
1,1
0,3
1,0
0,9
n.d.
0,10
0,036
0,07
0,05
0,028
n.d.
0,05
0,039
0,03
0,04
0,025
n.d.
-2
6
-69
-1
-9
n.d.
-90
-96,4
-93
-95
-97,2
n.d.
-94,8
-96,1
-96,5
-95,8
-97,5
n.s.
n.s.
+++
n.s.
n.s.
+++
+++
+++
+++
+++
+++
+++
+++
Glyc3P
Glyc
Ala
Ser
0,8
1,1
3,6
0,8
0,7
1,1
4,2
1,1
n.d.
n.d.
0,05
n.d.
n.d.
n.d.
0,07
n.d.
n.d.
n.d.
0,19
n.d.
n.d
n.d
0,23
n.d.
-16
12
257
-20
-28
7
324
8
n.d.
n.d.
-95
n.d.
n.d.
n.d
-93
n.d.
n.d.
n.d.
-81
n.d.
n.d.
n.d
-77
n.d.
n.s.
+++
+++
n.s.
+++
+
+++
n.s.
+++
+++
+++
+++
Cit
aKG
Succ
Fum
Mal
0,5
1,4
1,2
0,4
0,3
0,8
1,6
1,0
0,4
0,3
0,094
n.d.
n.d.
n.d.
n.d.
0,11
n.d.
n.d.
n.d.
n.d.
0,06
n.d.
n.d.
n.d.
n.d.
0,12
n.d
n.d
n.d
n.d
-49
40
17
-64
-72
-24
58
4
-64
-69
-90,6
n.d.
n.d.
n.d.
n.d.
-89
n.d
n.d
n.d
n.d
-94,1
n.d.
n.d.
n.d.
n.d.
-87,6
n.d
n.d
n.d
n.d
+++
+++
++
+++
+++
n.s.
+++
n.s.
+++
+++
+++
+++
+++
+++
18
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2-deoxyglucose using pulsed stable isotope-resolved
metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Table S 13 – Effect of 2-Deoxyglucose on the central carbon metabolism of T98G cells.
T98G cells were incubated with two different concentrations of 2-Deoxyglucose for 15 min. Rearrangement of metabolism were monitored by 13Cglucose incorporation for 3 minutes. Fold changes of peak intensity and 13C-label incorporation are shown relative to control. Two-sided,
heteroscedastic t-test indicates significant changes (+++ < 0.005, ++ < 0.01, + < 0.05, n.s. = not significant, n.d. = not detected).
2mM
2mM
10mM
-24
-49
-4.9
-6.3
-28
-53
n.s.
++
n.s. n.s.
+
+++
0.6
-27
-46
7.8
5.0
-20
-40
n.s.
n.s.
n.s. n.s.
n.s.
n.s.
Fru
1.1
1.0
0.9
0.8
1.0
0.8
7.9
-0.2
-10.6
-21.7
-3.3
-22.5
n.s.
n.s.
n.s. n.s.
n.s.
++
3PGA
0.8
n.d.
1.1
n.d.
0.9
n.d.
-16.9
n.d.
6.6
n.d.
-11.2
n.d.
n.s.
n.s.
n.s.
Pyr
0.7
0.3
1.3
0.8
0.9
0.2
-28.7 -71.1
31.3
-21.8
-6.8
-79.0
+
+++
+++ n.s.
n.s.
+++
Lac
0.8
0.7
1.2
0.8
1.0
0.6
-19.6 -28.5
20.2
-17.5
-4.7
-41.0
n.s. +++
n.s.
+
n.s.
+++
Glyc3P
0.9
1.1
1.8
1.3
1.7
1.6
-8.1
14.3
78.2
26.6
72.9
57.2
n.s.
n.s.
+
n.s.
n.s.
n.s.
Glyc
1.0
1.1
n.d. n.d.
n.d.
n.d.
-3.8
5.3
n.d.
n.d.
n.d.
n.d.
n.s.
n.s.
Ala
1.1
0.8
1.7
1.1
1.3
0.6
11.0
-15.8
70.2
5.3
31.9
-44.6
n.s.
n.s.
n.s. n.s.
n.s.
n.s.
Ser
0.8
0.7
n.d. n.d.
n.d.
n.d.
-17.7 -31.8
n.d.
n.d.
n.d.
n.d.
n.s.
+
n.s. n.s.
Cit
1.3
1.9
1.7
1.4
2.2
2.5
30.0
85.6
66.7
36.8
+++ n.s.
+++
+++
aKG
1.0
0.5
n.d. n.d.
n.d.
n.d
-0.1
-47.5
n.d.
n.d
n.d.
Succ
0.8
0.8
n.d. n.d.
n.d.
n.d
-17.3 -15.3
n.d.
n.d
n.d.
n.d
Fum
0.9
0.6
1.1
1.0
1.0
0.6
-6.2
-38.9
13.1
-2.9
3.1
-41.5
n.s. +++
n.s. n.s.
n.s.
n.s.
Mal
0.9
0.6
1.4
1.1
1.2
0.6
-7.2
-40.6
37.2
11.3
23.1
-35.8
n.s. +++
n.s. n.s.
n.s.
n.s.
n.d
n.s. +++
n.s.
++
+
+
Label
Inc.
10mM
10mM
0.5
0.8
115.4 152.6
Peak
intensity
2mM
0.7
1.0
10mM
0.9
1.1
2mM
Label Inc. x
intensity
10mM
Label
Inc.
2mM
1.0
0.5
10mM
0.5
0.7
2mM
0.8
F6P
10mM
G6P
2mM
10mM
Peak
intensity
Label
Inc. x
intensity
Compound
10mM
Label Inc.
x intensity
T-Test
Peak
intensity
2mM
Label
Inc.
% Change relative to control
2mM
Fold changes relative to control
19
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2deoxyglucose using pulsed stable isotope-resolved metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Figure S 6 - Time dependency of 13C-label incorporation into intermediates of the central carbon
metabolism.
HEK293 cells, grown in DMEM supplemented with 10 % FBS, 2.5 g/L glucose, and 4 mM glutamine,
were incubated with 13C-glucose up to 16 min. Harvest, metabolite extraction, GC-MS measurement
and determination of 13C-label incorporation were done as described in the Methods section.
20
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2-deoxyglucose using pulsed stable isotope-resolved
metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Figure S 7 – Graphical representation of small molecule inhibitor induced rearrangement of central carbon metabolism.
The incorporation of 13C-glucose and abundance of metabolites are presented by color or size relative to the untreated control after 15 minutes of
inhibitor treatment and 3 min of 13C-glucose incubation.
21
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2deoxyglucose using pulsed stable isotope-resolved metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Figure S 8 - Concentration dependency of BrPyr induced inhibition in carbon flow from 13Cglucose to lactate.
Three different concentrations of 3-Bromopyruvate were applied for 15 minutes to T98G cells
and incorporation of u-13C-glucose was monitored for 3 minutes in 3 biological replicates. Fold
change of incorporated label, intensity (peak area) and labeled intensity were plotted relative
to an untreated control.
22
Additional file 1
Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2deoxyglucose using pulsed stable isotope-resolved metabolomics
Pietzke and Zasada et al., Cancer and Metabolism 2014
Figure S 9 - Time and concentration dependent accumulation of 2-deoxyglucose-6-phosphate (2DGP). Shown are averages of ratios of two biological replicates measured in two technical replicates.
23