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Monitoring Metabolites Consumption and Secretion in Cultured Cells Using Ultra-Performance Liquid Chromatography Quadrupole-Time

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Monitoring Metabolites Consumption and Secretion in Cultured Cells Using Ultra-Performance Liquid Chromatography Quadrupole-Time Analytical and Bioanalytical Chemistry Electronic Supplementary Material Monitoring metabolites consumption and secretion in cultured cells using ultra-performance liquid chromatography quadrupole-time of flight mass spectrometry (UPLC-Q-ToF-MS) Giuseppe Paglia, Sigrún Hrafnsdóttir, Manuela Magnúsdóttir , Ronan MT Fleming, Steinunn Thorlacius, Bernhard Ø Palsson and Ines Thiele. 1 Figure S1. Overlay extracted ion chromatograms obtained using mobile phases at different pHs. 1) SDMA, 2) ADMA, 3) Arginine, 4) Lysine, 5) Ornithine, 6) Cystine, 7) 5-MTA, 8) Deoxyadenosine, 9) Hypoxanthine, 10) Adenosine, 11) Xanthine 12) Inosine, 13) Deoxyguanosine, 14) Guanine, 15) Guanosine. 2 9 (a) (d) 3 10 pH3 1 pH3 11 8 15 7 4 5 14 6 12 13 1,2 10 13,14 (b) (e) pH5 9 pH5 8 3 12 4 7 15 11 4 1 10 (c) 2 (f) pH9 9 pH9 3 8 13,14 4 11 7 12 3 2 Figure S2. Growth and apoptosis of Molt-4 and CCRF-CEM treated with AMPK activators. Cells were resuspended in RPMI advanced media containing 0.5 mM AICAR, 0.1 mM A-769662 or DMSO (0.7%) and cultured at 37°C and 5% CO2 in 24 well plates. Cells were counted using an automatic cell counter (Countess, Invitrogen). The graphs show fraction of viable cells (not stained with Trypan blue). Apoptosis was measured after 48 hrs using Annexin V binding and flow cytometry according to the manufactures instructions (Beckman Coulter). All apoptotic cells are shown, including cells undergoing necrosis (stained with Annexin V-PE and 7-AAD). Data from cells grown without DMSO and in RPMI, 10% FBS are also shown for comparison. Data shown represent the average and standard deviation of three independent experiments. Cells treated with the AMPK activators showed marked decrease in growth and increase in apoptosis. The concentration of viable cells was reduced by approximately 50% as compared to the controls after 48 hrs. Both AMPK activators increased the fraction of apoptotic cells for both cell lines, with Molt-4 showing more apoptosis. CCRF-CEM 45 Apototic cells 25 Molt-4 25 DMSO 40 DMSO 20 AICAR ) 35 5 20 ) AICAR CCRF-CEM 5 A769662 A769662 30 Molt-4 15 15 25 10 10 20 % apoptosis% 15 5 (10 Viablecells/ml 5 Viable cells/ml (10 Viablecells/ml 10 5 0 0 0 0 20 40 60 0 20 40 60 time (hr) 10% FBS RPMI adv DMSO AICAR A-769662 time (hr) 3 Figure S3. Total ATP in cells, treated as described for Fig. S2, was measured using a luminescent assay (CellTiter-Glo, Promega). Data were normalized to cell volume. For Molt-4 cells, concentrations were 6.1±0.7, 6.9±1.1 and 4.1±0.5 mM for DMSO, AICAR and A-769662, respectively. For CCRF-CEM cells, concentrations were 3.9±0.5, 9.1±0.8 and 3.5±0.3 mM for DMSO, AICAR and A-769662, respectively. Data shown represent average and standard deviation of three independent experiments. The total ATP, normalized to cell volume, was similar in cells treated with AMPK activators and control except in CCRF-CEM cells treated with AICAR, was it was increased 2 fold. ATP concentration 300 Molt-4 250 CCRF-CEM 200 150 100 50 ATP/cell volumecontrol)of (% ATP/cell 0 DMSO AICAR A769662 4 Figure S4. Metabolites secreted during the CCFR-CEM cells culture after 48 hours with AICAR treatments. The amount of compound secreted is given as log of pmoles/cells. Data from three biological replicates injected twice are shown. Concentration of carbamoyl-aspartate was calculated using dihydroorotic acid calibration curve. 5 Table S1. List of metabolites investigated during the method development. Neutral RT ESI + ESI - Metabolite Kegg ID Theoretical Observed Theoretical Observed Formula [min] ∆ ppm ∆ ppm mass mass mass mass 1 2-deoxyadenosine HMDB00101 C10H13N5O3 4.04 252.1097 252.1096 -0.4 - - - 2 2-deoxyguanosine HMDB00085 C10H13N5O4 6.01 268.1046 268.1065 7.1 266.0889 266.0883 -2.3 3 2-hydroxybutyric acid HMDB00008 C4H8O3 1.68 - - - 103.0395 103.04 4.9 4 2-ketobutyric acid HMDB00005 C4H6O3 1.26 - - - 101.0239 101.0236 -3 5 3-methyl-2-oxobutyrate HMDB00019 C5H8O3 1.14 - - - 115.0395 115.0393 -1.7 6 3-methyl-2-oxovalerate HMDB00491 C6H10O3 1.15 - - - 129.0552 129.0551 -0.8 7 4-hydroxyphenylpiruvate HMDB00707 C9H8O4 2.61 - - - 179.0344 179.0376 17.9 8 5-Methylthioadenosine (5-MTA) HMDB01173 C11H15N5O3S 3.33 298.0974 298.099 5.4 296.0817 296.0815 -0.7 9 A769962 - C20H12N2O3S 3.89 361.0647 361.064 -1.9 359.049 359.0507 4.7 10 Acetylalanine HMDB00766 C5H9NO3 2.1 132.0661 132.0671 7.6 130.0504 130.0516 9.2 11 Acetylmethionine HMDB11745 C7H13NO3S 1.86 192.0694 192.0699 2.6 190.0538 190.0555 8.9 12 Adenine HMDB00034 C5H5N5 4.04 136.0623 136.0625 1.5 134.0467 134.0469 1.5 13 Adenosine HMDB00050 C10H13N5O4 4.61 268.1046 268.1049 1.1 266.0889 266.0895 2.3 14 ADMA HMDB03334 C8H18N4O2 8.89 203.1508 203.1511 1.5 201.1352 201.1357 2.5 15 AICAR - C9H14N4O5 5.16 259.1042 259.105 3.1 257.0886 257.0898 4.7 16 Allantoin HMDB00462 C4H6N4O3 4.9 - - - 157.0362 157.0372 6.4 17 Alanine HMDB00161 C3H7NO2 6.61 90.0555 90.056 5.6 88.0399 88.0396 -3.4 18 Alanyl-Glutamine - C8H15N3O4 7.42 218.1141 218.1147 2.8 216.0984 216.0982 -0.9 19 Arabinose HMDB11734 C5H10O5 5.07 - - - 149.045 149.0444 -4 20 Arginine HMDB00517 C6H14N4O2 10.41 175.1195 175.1201 3.4 173.1039 173.1038 -0.6 21 Asparagine HMDB00168 C4H8N2O3 6.14 133.0613 133.0602 -8.3 - - - 22 Aspartic Acid HMDB00191 C4H7NO4 8.69 134.0453 134.0456 2.2 132.0297 132.0302 3.8 23 Biotin HMDB00030 C10H16N2O3S 3.16 245.096 245.0978 7.3 243.0803 243.08 -1.2 24 Choline HMDB00097 C5H14NO 1.62 104.1075 104.1082 6.7 - - - 25 Citrate HMDB00094 C6H8O7 8.28 - - - 191.0192 191.0193 0.5 26 Cysteine HMDB00574 C3H7NO2S 13.17 122.0276 122.028 3.3 120.0119 120.0129 8.3 27 Cystine HMDB00192 C6H12N2O4S2 12.37 241.0317 241.0318 0.4 239.016 239.0164 1.7 28 Dihydroorotic acid HMDB03349 C5H6N2O4 3.74 159.0406 159.0411 3.1 157.0249 157.0258 5.7 6 Table S1 (continued) 29 Dimethylglycine HMDB00092 C4H9NO2 6.29 104.0712 104.0718 5.8 102.0555 102.0556 1 30 Folic Acid HMDB00121 C19H19N7O6 9.13 - - - 440.1319 440.1312 -1.6 31 Fructose* HMDB00660 C6H12O6 5.8 203.0532 203.0537 2.5 179.0556 179.0552 -2.2 32 Galactose* HMDB00143 C6H12O6 7.62 203.0532 203.0541 4.4 179.0556 179.0568 6.7 33 Glucose* HMDB00122 C6H12O6 7.29 203.0532 203.0534 1 179.0556 179.0558 1.1 34 Glutamic Acid HMDB00148 C5H9NO4 7.98 148.061 148.0613 2 146.0453 146.046 4.8 35 Glutamine HMDB00641 C5H10N2O3 8.65 147.077 147.0776 4.1 145.0613 145.0617 2.8 36 Glycine HMDB00123 C2H5NO2 7.25 76.0399 76.0405 7.9 74.0242 74.0245 4.1 37 Guanine HMDB00132 C5H5N5O 6.15 152.0572 152.0583 7.2 150.0416 150.0411 -3.3 38 Guanosine HMDB00133 C10H13N5O5 6.86 284.0995 284.0998 1.1 282.0838 282.0835 -1.1 39 Hippuric Acid HMDB00714 C9H9NO3 1.79 180.0661 180.0665 2.2 178.0504 178.052 9 40 Histidine HMDB00177 C6H9N3O2 10.63 156.0773 156.0773 0 154.0617 154.0614 -1.9 41 Hydroxyproline HMDB00725 C5H9NO3 7.91 132.0661 132.0664 2.3 130.0504 130.0516 9.2 42 Hypoxanthine HMDB00157 C5H4N4O 4.28 137.0463 137.0466 2.2 135.0307 135.0312 3.7 43 Inosine HMDB00195 C10H12N4O5 5.39 269.0886 269.0892 2.2 267.0729 267.0736 2.6 44 Inositol* HMDB00211 C6H12O6 9.92 203.0532 203.0537 2.5 179.0556 179.0563 3.9 45 Isoleucine HMDB00172 C6H13NO2 5.67 132.1025 132.1031 4.5 130.0868 130.0878 7.7 46 Lactate HMDB00190 C3H6O3 1.93 - - - 89.0239 89.0247 9 47 Lactose* HMDB00186 C12H22O11 10.35 365.106 365.1071 3 341.1084 341.1079 -1.5 48 Leucine HMDB00687 C6H13NO2 5.81 132.1025 132.1032 5.3 130.0868 130.0872 3.1 49 Lysine HMDB00182 C6H14N2O2 11.05 147.1134 147.1113 -14.3 145.0977 145.098 2.1 50 Malate HMDB00744 C4H6O5 2.97 - - - 133.0137 133.0141 3 51 Maltose* HMDB00163 C12H22O11 10.55 365.106 365.1057 -0.8 341.1084 341.1087 0.9 52 Mannose* HMDB01163 C6H12O6 6.47 203.0532 203.0539 3.4 179.0556 179.0552 -2.2 53 Methionine HMDB00696 C5H11NO2S 6.12 150.0589 150.0601 8 148.0432 148.043 -1.4 54 Nicotinamide HMDB01406 C6H6N2O 1.99 123.0558 123.0564 4.9 - - - 55 Ornithine HMDB00214 C5H12N2O2 11 133.0977 133.0974 -2.3 131.0821 131.0825 3.1 56 Orotate HMDB00226 C5H4N2O4 6.24 157.0249 157.0255 3.8 155.0093 155.009 -1.9 57 Oxoproline HMDB00267 C5H7NO3 2.89 130.0504 130.051 4.6 128.0348 128.0352 3.1 58 p-Aminobenzoic acid (PABA) HMDB01392 C7H7NO2 1.31 138.0555 138.0562 5.1 - - - 59 Panthothenate HMDB00210 C9H17NO5 2.74 220.1185 220.1191 2.7 218.1028 218.1031 1.4 60 Phenol red - C19H14O5S 4.43 355.064 355.0645 1.4 353.0484 353.0497 3.7 61 Phenylalanine HMDB00159 C9H11NO2 5.97 166.0868 166.0878 6 164.0712 164.0705 -4.3 62 Proline HMDB00162 C5H9NO2 6.31 116.0712 116.0714 1.7 - - - 7 Table S1 (continued) 63 Pyridoxal HMDB01545 C8H9NO3 3.25 168.0661 168.0667 3.6 166.0504 166.0506 1.2 64 Pyridoxamine HMDB01431 C8H12N2O2 6.95 169.0977 169.0978 0.6 167.0821 167.0817 -2.4 65 Pyridoxate HMDB00017 C8H9NO4 4.4 184.061 184.0613 1.6 182.0453 182.0459 3.3 66 Pyridoxine HMDB00239 C8H11NO3 4.26 170.0817 170.0819 1.2 168.0661 168.0666 3 67 Pyruvate HMDB00243 C3H4O3 1.35 - - - 87.0082 87.0089 8 68 Raffinose* HMDB03213 C18H32O16 12.37 527.1588 527.1594 1.1 503.1612 503.1632 4 69 Riboflavin HMDB00244 C17H20N4O6 5.53 377.1461 377.1466 1.3 375.1305 375.1313 2.1 70 S-Adenosylhomocysteine (SAH) HMDB04145 C14H20N6O5S 10.01 385.1294 385.1311 4.4 383.1138 383.114 0.5 71 SDMA HMDB01539 C8H18N4O2 8.72 203.1508 203.1513 2.5 201.1352 201.1357 2.5 72 Serine HMDB00187 C3H7NO3 8.68 106.0504 106.0508 3.8 104.0348 104.035 1.9 73 Succinate HMDB00254 C4H6O4 2.46 - - - 117.0188 117.0196 6.8 74 Sucrose* HMDB00258 C12H22O11 10.11 365.106 365.107 2.7 341.1084 341.1089 1.5 75 Thiamine HMDB00235 C12H17N4OS 5.98 265.1123 265.1137 5.3 - - - 76 Threonine HMDB00167 C4H9NO3 7.74 120.0661 120.0666 4.2 118.0504 118.051 5.1 77 Thymine HMDB00262 C5H6N2O2 2.45 127.0508 127.0511 2.4 125.0351
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