Supplementary Information for the Article

Supplementary Information for the Article Separation and quantitation of water soluble cellular metabolites by hydrophilic interaction chromatography tandem spectrometry Sunil U. Bajad a, Wenyun Lu a, Elizabeth H. Kimball a, Jie Yuan a, Celeste Peterson b, and Joshua D. Rabinowitz a * a LewisSigler Institute for Integrative Genomics and Department of Chemistry Princeton University, Princeton, NJ 08544, USA b Department of Molecular Biology Princeton University, Princeton, NJ 08544, USA The main manuscript text focuses on the metabolome as a whole and, to a lesser extent, on compounds of specific interest. Here, in the Supplementary Information, we provide comprehensive results for every individual compound studied, in the form of three tables: Table S1: Known isomers of the metabolites investigated here (begins on Page S2) Table S2: MS/MS parameters and LCMS/MS method performance for compound standards (begins on Page S4) Table S3: Performance of different chromatography conditions for distinct cellular metabolites (begins on Page S12) Bajad et al., 2006 Supplementary Materials Page S1 of 17

Table S1: Known isomers of the metabolites investigated here *. The table is limited to those isomers that are known components of metabolism, as indicated by their being contained in the Ecocyc database (www.ecocyc.org). Metabolite ion formula Isomers Alanine C 3 H 8 NO 2 90 βalanine, DAlanine, Sarcosine Acetoacetate C 4 H 5 O 3 101 2Oxobutanoate, Succinate semialdehyde Serine C 3 H 8 NO 3 106 DSerine Fumarate C 4 H 3 O 4 115 Maleate Succinate C 4 H 5 O 4 117 Methylmalonate Threonine a C 4 H 8 NO 3 118 LAllothreonine, Homoserine, DThreonine Valine C 5 H 12 NO 2 118 Betaine Homoserine a C 4 H 10 NO 3 120 LThreonine, Allothreonine, LAllothreonine, DThreonine Cysteine C 3 H 8 NO 2 S 122 DCysteine Nicotinate C 6 H 4 NO 2 122 Picolinate Aspartate C 4 H 6 NO 4 132 DAspartate (Iso)leucine* C 6 H 14 NO 2 132 LIsoleucine, LLeucine Malate C 4 H 5 O 5 133 (R)Malate paminobenzoate/anthranilate* C 7 H 6 NO 2 136 paminobenzoate, Anthranilate phydroxybenzoate C 7 H 5 O 3 137 Salicylate Acetylphosphate C 2 H 4 O 5 P 139 Phosphonoacetate Glutamine C 5 H 11 N 2 O 3 147 Glutamide Glutamate C 5 H 10 NO 4 148 OAcetylLserine, Dglutamate Carnitine C 7 H 16 NO 3 162 DCarnitine Phenylpyruvate C 9 H 7 O 3 163 4Coumarate Dihydroxyacetonephosphate C 3 H 6 O 6 P 169 DGlyceraldehyde3phosphate Aconitate* C 6 H 5 O 6 173 cisaconitate, transaconitate myoinositol C 6 H 11 O 6 179 Mannose, αdgalactose, 2Deoxygluconate, DAllose, Fructose, βdglucose, βdgalactose 3Phosphoglycerate C 3 H 6 O 7 P 185 2Phosphoglycerate Citrate C 6 H 7 O 7 191 2,5DiketoDgluconate, 5Keto4deoxyDglucarate, Isocitrate, 2,3DiketoLgulonate 2DehydroDgluconate C 6 H 9 O 7 193 5Ketogluconate, Galacturonate, DTagaturonate, Fructuronate, Glucuronate, 3KetoLgulonate, 2 KetoLgulonate Gluconate C 6 H 11 O 7 195 DMannonate, LIdonate, DGalactonate, DAltronate DGlucarate C 6 H 9 O 8 209 DGalactarate Deoxyribosephosphate* C 5 H 10 O 7 P 213 Deoxyribose1phosphate, Deoxyribose5phosphate, 1DeoxyDxylulose 5phosphate Prephenate C 10 H 9 O 6 225 Chorismate, Isochorismate Bajad et al., 2006 Supplementary Materials Page S2 of 17

ion formula Metabolite Isomers DRibulose5phosphate, DRibose5phosphate, DRibulose1phosphate, Ribose1phosphate, DRib(ul)ose5phosphate* C 5 H 10 O 8 P 229 DArabinose5phosphate, Xylulose5phosphate, LRibulose5phosphate, LXylulose5phosphate Deoxyadenosine C 10 H 14 N 5 O 3 252 5'Deoxyadenosine αdglucose1phosphate, DFructose6phosphate, Mannose6phosphate, Mannose1 phosphate, αdgalactose1phosphate, αdglucose6phosphate, βdglucose6phosphate, β DGlucose 1phosphate, Fructose1phosphate, 1LmyoInositol 1phosphate, 1DmyoInositol 1 DHexosephosphate C 6 H 12 O 9 P 259 phosphate, Tagatose6phosphate, DAllose6phosphate Glucosamine1P b C 6 H 15 NO 8 P 260 Glucosamine6P Glucosamine6P b C 6 H 15 NO 8 P 260 Glucosamine1P Adenosine c C 10 H 14 N 5 O 4 268 Deoxyguanosine Deoxyguanosine c C 10 H 14 N 5 O 4 268 Adenosine NAcetylglucosamine1 phosphate C 8 H 15 NO 9 P 300 NAcetylDglucosamine6phosphate, NAcetylDmannosamine6phosphate Fructose1,6bisphosphate C 6 H 13 O 12 P 2 339 Glucose1,6bisphosphate, Tagatose1,6bisphosphate Trehalose C 12 H 21 O 11 341 Sucrose, Lactose, Maltose, Cellobiose, Melibiose AMP d C 10 H 15 N 5 O 7 P 348 dgmp dgmp d C 10 H 15 N 5 O 7 P 348 AMP ADP e C 10 H 14 N 5 O 10 P 2 426 3',5'ADP, dgdp dgdp e C 10 H 14 N 5 O 10 P 2 426 ADP, 3',5'ADP ATP f C 10 H 15 N 5 O 13 P 3 506 dgtp dgtp f C 10 H 15 N 5 O 13 P 3 506 ATP UDPDglucose C 15 H 23 N 2 O 17 P 2 565 UDPgalactose, UDPDgalacto1,4furanose ADPDglucose C 16 H 24 N 5 O 15 P 2 588 Guanosine diphosphate fucose UDPNacetylDglucosamine C 17 H 26 N 3 O 17 P 2 606 UDPNacetylDmannosamine SuccinylCoA C 25 H 41 N 7 O 19 P 3 S 868 LMethylmalonylCoA, DMethylmalonylCoA * Collective name assigned to describe isomers that were explicitly studied and could not be differentiated by product ion or retention time; the studied compounds are marked in bold in the isomer column a Compounds form distinct product ions b Compounds form distinct products and have different retention times c Compounds form distinct products and have different retention times d Compounds form distinct product ions e Compounds have different retention times f Compounds have different retention times Bajad et al., 2006 Supplementary Materials Page S3 of 17

Table S2: MS/MS parameters and LCMS/MS method performance for compound standards. Metabolite ion formula* Collision energy (ev) ion Stability LOD formula (ng/ml) RT (min) R 2 Intraday %RSD Interday % RSD Glycine C 2 H 6 NO 2 76 78 16 30 31 CH 4 N 50 13.5 0.999 7 14 Putrescine C 4 H 13 N 2 89 93 11 72 76 C 4 H 10 N >2000 n/a n/a n/a n/a Alanine C 3 H 8 NO 2 90 93 11 44 46 C 2 H 6 N 10 13 0.993 10 10 Acetoacetate C 4 H 5 O 3 101 105 21 57 60 C 3 H 5 O 100 13.4 n/a n/a n/a Choline C 5 H 14 NO 104 109 19 60 63 C 3 H 10 N 1 13.4 0.994 7 6 Glycerate C 3 H 5 O 4 105 108 15 75 77 C 2 H 3 O 3 5 15.4 0.963 3 29 Serine C 3 H 8 NO 3 106 109 13 60 62 C 2 H 6 NO 5 14 0.989 19 5 Cytosine C 4 H 6 N 3 O 112 116 17 95 99 C 4 H 3 N 2 O 5 10.6 0.995 17 7 Uracil C 4 H 5 N 2 O 2 113 117 23 70 73 C 3 H 4 NO >2000 n/a n/a n/a n/a Fumarate C 4 H 3 O 4 115 119 11 71 74 C 3 H 3 O 2 500 24.7 0.964 4 29 Proline C 5 H 10 NO 2 116 121 11 70 74 C 4 H 8 N 5 12.7 0.99 15 10 Succinate C 4 H 5 O 4 117 121 10 73 76 C 3 H 5 O 2 1000 23.7 n/a 5 10 Threonine C 4 H 8 NO 3 118 122 12 74 76 C 2 H 4 NO 2 5 13.4 0.992 10 9 Valine C 5 H 12 NO 2 118 123 11 55 59 C 4 H 7 5 12.1 0.996 17 11 Homoserine C 4 H 10 NO 3 120 124 30 44 46 C 2 H 6 N 50 13.1 0.999 20 9 Cysteine C 3 H 8 NO 2 S 122 125 27 59 61 C 2 H 3 S >2000 n/a n/a n/a n/a Nicotinate C 6 H 4 NO 2 122 128 14 78 83 C 5 H 4 N 10 14.3 0.994 16 21 Nicotinamide C 6 H 7 N 2 O 123 129 20 80 85 C 5 H 6 N 5 7.3 0.987 b 26 b 10 b Taurine C 2 H 6 NO 3 S 124 126 16 80 80 SO 3 10 13.2 0.998 10 9 Thymine C 5 H 7 N 2 O 2 127 132 17 110 115 C 5 H 4 NO 2 500 7.4 0.917 16 9 Agmatine C 5 H 15 N 4 131 136 16 72 76 C 4 H 10 N >2000 n/a n/a n/a n/a Oxaloacetate C 4 H 3 O 5 131 135 12 87 90 C 3 H 3 O 3 1000 24.9 n/a n/a n/a Bajad et al., 2006 Supplementary Materials Page S4 of 17

Metabolite ion formula* Collision energy (ev) ion Stability LOD formula (ng/ml) RT (min) R 2 Intraday %RSD Interday % RSD Aspartate C 4 H 6 NO 4 (Iso)leucine C 6 H 14 NO 2 Asparagine C 4 H 9 N 2 O 3 Malate C 4 H 5 O 5 Ornithine C 5 H 13 N 2 O 2 Adenine C 5 H 6 N 5 132 136 17 88 91 C 3 H 6 NO 2 10 17.2 0.957 5 7 132 138 11 86 91 C 5 H 12 N 1 11.5 0.992 20 12 133 137 17 74 76 C 2 H 4 NO 2 133 137 12 115 119 C 4 H 3 O 4 25 13.8 0.994 8 8 10 23.6 0.993 14 5 133 138 12 70 74 C 4 H 8 N 5 14.9 0.993 9 5 136 141 24 119 124 C 5 H 3 N 4 1 11 0.994 15 5 Homocysteine C 4 H 10 NO 2 S 136 140 10 90 93 C 3 H 8 N 5 25 12.8 0.957 16 6 paminobenzoate/ Anthranilate C 7 H 6 NO 2 136 143 16 92 98 C 6 H 6 N 250 17.5 0.982 b 21 b 10 b Hypoxanthine C 5 H 5 N 4 O 137 142 19 110 114 C 4 H 4 N 3 O 10 13.6 0.984 18 28 phydroxybenzoate C 7 H 5 O 3 Acetylphosphate C 2 H 4 O 5 P 139 141 22 79 79 PO 3 Carbamoylphosphate CH 3 NO 5 P 140 141 22 79 79 PO 3 137 144 21 93 99 C 6 H 5 O 50 24.9 0.994 b 7 b 11 b Histidinol C 6 H 12 N 3 O 142 148 18 95 100 C 5 H 7 N 2 αketoglutarate C 5 H 5 O 5 Spermidine C 7 H 20 N 3 Glutamine C 5 H 11 N 2 O 3 Lysine C 6 H 15 N 2 O 2 Glutamate C 5 H 10 NO 4 145 150 11 101 105 C 4 H 5 O 3 50 23.4 n/a n/a n/a 100 24.2 n/a n/a n/a 5 11.7 0.996 10 7 100 24.8 0.993 13 6 146 153 13 112 119 C 7 H 14 N >2000 n/a n/a n/a n/a 147 152 15 84 88 C 4 H 6 NO 5 13.5 0.992 11 7 147 153 15 84 89 C 5 H 10 N 50 14.8 0.982 6 6 148 153 15 84 88 C 4 H 6 NO 10 16.9 0.991 6 3 Methionine C 5 H 12 NO 2 S 150 155 10 133 138 C 5 H 9 O 2 S 5 12.2 0.995 6 11 Guanine C 5 H 6 N 5 O 152 157 18 110 114 C 4 H 4 N 3 O 10 12.1 0.99 33 11 2,3Dihydroxybenzoate C 7 H 5 O 4 153 160 17 109 115 C 6 H 5 O 2 1 16 0.994 1 14 Xanthine C 5 H 5 N 4 O 2 153 158 16 110 114 C 4 H 4 N 3 O >2000 n/a n/a n/a n/a Orotate C 5 H 3 N 2 O 4 155 160 13 111 115 C 4 H 3 N 2 O 2 1 16.6 0.988 2 5 Histidine C 6 H 10 N 3 O 2 156 162 12 110 115 C 5 H 8 N 3 1 14 0.989 7 12 Bajad et al., 2006 Supplementary Materials Page S5 of 17

Metabolite ion formula* Collision energy (ev) ion Stability LOD formula (ng/ml) RT (min) R 2 Intraday %RSD Interday % RSD Dihydroorotate C 5 H 5 N 2 O 4 157 162 12 113 117 C 4 H 5 N 2 O 2 5 14.2 0.994 6 2 Allantoin C 4 H 7 N 4 O 3 159 163 11 99 102 C 3 H 3 N 2 O 2 >2000 n/a n/a n/a n/a Carnitine C 7 H 16 NO 3 162 169 18 103 107 C 4 H 7 O 3 1 12.3 0.994 10 23 Phenylpyruvate C 9 H 7 O 3 163 172 11 91 98 C 7 H 7 5 11.3 0.996 15 7 Phenylalanine C 9 H 12 NO 2 166 175 28 103 111 C 8 H 7 1 11.7 0.991 16 6 Quinolinate C 7 H 4 NO 4 166 173 12 122 128 C 6 H 4 NO 2 5 24.7 0.993 10 19 Phosphoenolpyruvate C 3 H 4 O 6 P 167 170 41 79 79 PO 3 50 25 0.956 33 14 Dihydroxyacetonephosphate C 3 H 6 O 6 P 169 172 38 79 79 PO 3 1500 24.6 n/a n/a n/a Pyridoxine C 8 H 12 NO 3 170 178 22 134 142 C 8 H 8 NO 1 12.1 0.984 10 20 Glycerol3phosphate C 3 H 8 O 6 P 171 174 13 79 79 PO 3 500 23.4 0.857 5 10 Arginine C 6 H 13 N 4 O 2 173 179 12 131 136 C 5 H 11 N 2 O 2 10 13.9 0.995 6 13 Shikimate C 7 H 9 O 5 173 180 18 93 99 C 6 H 5 O 100 16.7 0.999 9 24 Aconitate C 6 H 5 O 6 Citrulline C 6 H 12 N 3 O 3 Allantoate C 4 H 7 N 4 O 4 CarbamoylLaspartate C 5 H 7 N 2 O 5 NαAcetylornithine C 7 H 15 N 2 O 3 Hydroxyphenylpyruvate C 9 H 7 O 4 myoinositol C 6 H 11 O 6 Glucosamine C 6 H 14 NO 5 173 179 10 129 134 C 5 H 5 O 4 174 180 13 131 136 C 5 H 11 N 2 O 2 175 179 12 132 135 C 3 H 6 N 3 O 3 175 180 12 132 136 C 4 H 6 NO 4 175 182 14 115 120 C 5 H 9 NO 2 a 5 25 0.99 17 18 5 13.3 0.998 7 4 a 50 16.7 0.98 1 13 25 23.7 0.964 8 10 5 13.4 0.994 6 10 179 188 11 107 114 C 7 H 7 O 100 15.8 0.99 6 17 179 185 15 161 167 C 6 H 9 O 5 180 186 10 162 168 C 6 H 12 NO 4 Homocysteic acid C 4 H 8 NO 5 S 182 186 21 80 80 SO 3 Tyrosine C 9 H 12 NO 3 182 191 37 77 83 C 6 H 5 3Phosphoglycerate C 3 H 6 O 7 P 185 188 15 97 97 H 2 PO 4 3Phosphoserine C 3 H 9 NO 6 P 186 189 10 88 91 C 3 H 6 NO 2 100 12.4 0.929 24 19 10 11.4 0.991 3 17 >2000 n/a n/a n/a n/a 10 13.2 0.988 15 5 100 25 0.949 24 16 10 24 0.993 15 8 Bajad et al., 2006 Supplementary Materials Page S6 of 17

Metabolite ion formula* Collision energy (ev) ion Stability LOD formula (ng/ml) RT (min) R 2 Intraday %RSD Interday % RSD Citrate C 6 H 7 O 7 191 197 13 111 116 C 5 H 3 O 3 10 25 0.995 6 12 2DehydroDgluconate C 6 H 9 O 7 193 199 12 103 107 C 4 H 7 O 3 1 16.5 0.953 5 15 Gluconate C 6 H 11 O 7 195 201 15 129 134 C 5 H 5 O 4 a 5 16.2 0.958 4 16 Erythrose4phosphate C 4 H 8 O 7 P 199 203 17 97 97 H 2 PO 4 1000 22.8 n/a n/a n/a Tryptophan C 11 H 13 N 2 O 2 205 216 16 146 155 C 9 H 8 NO 5 12 0.995 11 6 DGlucarate C 6 H 9 O 8 209 215 16 85 89 C 4 H 5 O 2 250 23.5 0.944 17 11 Deoxyribosephosphate C 5 H 10 O 7 P 213 218 33 79 79 PO 3 1500 23.6 n/a n/a n/a Pantothenate C 9 H 16 NO 5 218 227 19 146 153 C 7 H 16 NO 2 1 14 0.998 8 8 Cystathionine C 7 H 15 N 2 O 4 S 223 230 11 134 138 C 4 H 8 NO 2 S 5 16.4 0.978 12 4 Prephenate C 10 H 9 O 6 225 235 15 91 97 C 6 H 3 O 100 24.5 n/a n/a n/a Deoxyuridine C 9 H 13 N 2 O 5 229 238 11 113 117 C 4 H 5 N 2 O 2 100 11.1 0.969 14 7 DRib(ul)ose5phosphate C 5 H 10 O 8 P 229 234 48 79 79 PO 3 a 100 24 0.995 20 20 Thymidine C 10 H 13 N 2 O 5 241 251 15 125 130 C 5 H 5 N 2 O 2 5 10.6 0.997 12 9 Uridine C 9 H 11 N 2 O 6 243 252 19 200 208 C 8 H 10 NO 5 10 10.6 0.986 12 9 Cytidine C 9 H 14 N 3 O 5 244 253 12 112 116 C 4 H 6 N 3 O 1 11.4 0.991 6 7 Biotin C 10 H 17 N 2 O 3 S 245 255 18 227 237 C 10 H 15 N 2 O 2 S 10 14.6 0.986 15 8 Deoxyadenosine C 10 H 14 N 5 O 3 252 262 20 136 141 C 5 H 6 N 5 <1 9.1 0.988 18 13 Deoxyinosine C 10 H 13 N 4 O 4 253 263 12 137 142 C 5 H 5 N 4 O 5 12.7 0.995 20 9 DHexosephosphate C 6 H 12 O 9 P 259 265 40 79 79 PO 3 500 23.3 0.943 8 4 Glucosamine1P C 6 H 15 NO 8 P 260 266 15 162 168 C 6 H 12 NO 4 100 22 0.993 7 8 Glucosamine6P C 6 H 15 NO 8 P 260 266 15 126 132 C 6 H 8 NO 2 100 23.4 0.978 16 8 Thiamine C 12 H 17 N 4 OS 265 277 17 122 128 C 6 H 8 N 3 1 10.8 0.994 12 15 Adenosine C 10 H 14 N 5 O 4 268 278 27 136 141 C 5 H 6 N 5 1 10.5 0.992 9 7 Deoxyguanosine C 10 H 14 N 5 O 4 268 278 15 152 157 C 5 H 6 N 5 O 1 12.1 0.995 9 5 Bajad et al., 2006 Supplementary Materials Page S7 of 17

Metabolite ion formula* Collision energy (ev) ion Stability LOD formula (ng/ml) RT (min) R 2 Intraday %RSD Interday % RSD Inosine C 10 H 13 N 4 O 5 6PhosphoDgluconate C 6 H 12 O 10 P 275 281 11 97 97 H 2 PO 4 Guanosine C 10 H 14 N 5 O 5 269 279 14 137 142 C 5 H 5 N 4 O 5 13.6 0.992 19 3 100 24.7 0.989 24 10 284 294 33 135 140 C 5 H 3 N 4 O 5 13.1 0.992 11 5 Xanthosine C 10 H 13 N 4 O 6 285 295 20 153 158 C 5 H 5 N 4 O 2 NAcetylglucosamine1 phosphate C 8 H 15 NO 9 P 300 308 32 79 79 PO 3 5 18.5 0.987 2 10 100 22.9 0.987 6 13 dump C 9 H 12 N 2 O 8 P 307 316 16 195 200 C 5 H 8 O 6 P 50 24.7 0.995 15 19 dcmp C 9 H 15 N 3 O 7 P 308 317 16 112 116 C 4 H 6 N 3 O 25 24.4 0.998 6 6 Reduced glutathione C 10 H 18 N 3 O 6 S 308 318 19 162 167 C 5 H 8 NO 3 S 100 20.4 0.999 13 13 GeranylPP C 10 H 19 O 7 P 2 313 323 18 79 79 PO 3 a 25 25.9 0.98 14 20 dtmp C 10 H 16 N 2 O 8 P 323 333 17 81 86 C 5 H 5 O 10 24.6 0.998 10 4 CMP C 9 H 15 N 3 O 8 P 324 333 16 112 116 C 4 H 6 N 3 O 50 24.3 0.981 4 14 UMP C 9 H 14 N 2 O 9 P 325 334 12 97 102 C 5 H 5 O 2 50 25 0.967 16 15 cyclicamp C 10 H 11 N 5 O 6 P 328 338 31 134 139 C 5 H 4 N 5 50 15 0.984 8 15 damp C 10 H 15 N 5 O 6 P 332 342 21 136 141 C 5 H 6 N 5 5 25 0.993 8 1 Fructose1,6bisphosphate C 6 H 13 O 12 P 2 339 345 28 97 97 H 2 PO 4 100 25.1 0.995 24 14 Trehalose C 12 H 21 O 11 341 353 18 179 185 C 6 H 11 O 6 25 12.5 0.987 13 20 Thiaminephosphate C 12 H 18 N 4 O 4 PS 345 357 13 122 128 C 6 H 8 N 3 25 17 0.99 8 8 AMP C 10 H 15 N 5 O 7 P 348 358 21 136 141 C 5 H 6 N 5 25 25.1 0.986 11 5 dgmp C 10 H 15 N 5 O 7 P 348 358 36 135 140 C 5 H 3 N 4 O 50 25.1 0.989 9 8 IMP C 10 H 14 N 4 O 8 P 349 359 19 137 142 C 5 H 5 N 4 O 10 25.1 0.995 9 8 GMP C 10 H 15 N 5 O 8 P 364 374 19 152 157 C 5 H 6 N 5 O 10 25.1 0.997 7 9 Xanthosine5phosphate C 10 H 14 N 4 O 9 P 365 375 11 97 102 C 5 H 5 O 2 25 25.2 0.989 10 8 Orotidinephosphate C 10 H 12 N 2 O 11 P 367 377 16 323 332 C 9 H 12 N 2 O 9 P 50 25 0.999 30 22 Riboflavin C 17 H 21 N 4 O 6 377 394 24 243 255 C 12 H 12 N 4 O 2 1 9.9 0.993 8 11 Bajad et al., 2006 Supplementary Materials Page S8 of 17

Metabolite ion formula* Collision energy (ev) ion Stability LOD formula (ng/ml) RT (min) R 2 Intraday %RSD Interday % RSD trans, transfarnesyl diphosphate C 15 H 27 O 7 P 2 381 396 21 79 79 PO 3 SAdenosylLhomocysteine C 14 H 21 N 6 O 5 S 385 399 19 136 141 C 5 H 6 N 5 dcdp C 9 H 14 N 3 O 10 P 2 PRPP C 5 H 12 O 14 P 3 386 395 25 159 159 HO 6 P 2 389 394 18 291 296 C 5 H 9 O 10 P 2 SAdenosylLmethionine C 15 H 23 N 6 O 5 S 399 414 13 250 260 C 10 H 12 N 5 O 3 dtdp C 10 H 15 N 2 O 11 P 2 CDP C 9 H 14 N 3 O 11 P 2 UDP C 9 H 13 N 2 O 12 P 2 ADP C 10 H 14 N 5 O 10 P 2 401 411 29 159 159 HO 6 P 2 402 411 29 159 159 HO 6 P 2 a 25 45.8 0.914 19 27 5 14 0.99 19 11 100 25 0.993 26 22 a 100 27.5 0.918 5 22 10 13.3 0.98 5 8 100 25 0.961 26 33 100 25 0.992 21 23 403 412 15 305 314 C 9 H 10 N 2 O 8 P 100 25.1 0.909 32 18 426 436 24 134 139 C 5 H 4 N 5 100 25.3 0.985 14 27 APS C 10 H 13 N 5 O 10 PS 426 436 20 346 356 C 10 H 13 N 5 O 7 P a 10 25 0.991 10 17 dgdp C 10 H 14 N 5 O 10 P 2 426 436 25 159 159 HO 6 P 2 100 27 0.994 14 18 IDP C 10 H 13 N 4 O 11 P 2 427 437 14 159 159 HO 6 P 2 1500 26.9 n/a n/a n/a Folate C 19 H 20 N 7 O 6 442 461 16 295 309 C 14 H 11 N 6 O 2 10 28 0.916 50 38 GDP C 10 H 14 N 5 O 11 P 2 7,8Dihydrofolate C 19 H 22 N 7 O 6 442 452 19 159 159 HO 6 P 2 100 27.1 0.923 b 8 b 9 b 444 463 30 178 185 C 7 H 8 N 5 O 25 27 n/a n/a n/a FMN C 17 H 20 N 4 O 9 P 455 472 19 213 218 C 5 H 10 O 7 P 25 25 0.989 9 26 5MethylTHF C 20 H 26 N 7 O 6 460 480 19 313 328 C 15 H 17 N 6 O 2 a 5 24.9 0.997 7 11 dctp C 9 H 15 N 3 O 13 P 3 466 475 28 159 159 HO 6 P 2 500 27.2 0.977 25 31 dutp C 9 H 14 N 2 O 14 P 3 467 476 25 159 159 HO 6 P 2 100 27.8 0.983 9 23 TTP C 10 H 16 N 2 O 14 P 3 481 491 31 159 159 HO 6 P 2 100 27.5 0.974 11 15 CTP C 9 H 15 N 3 O 14 P 3 482 491 32 159 159 HO 6 P 2 100 27.2 0.973 13 14 UTP C 9 H 14 N 2 O 15 P 3 483 492 33 159 159 HO 6 P 2 100 27.8 0.985 24 23 datp C 10 H 15 N 5 O 12 P 3 490 500 27 159 159 HO 6 P 2 100 29 0.927 17 26 Bajad et al., 2006 Supplementary Materials Page S9 of 17

Metabolite ion formula* Collision energy (ev) ion Stability LOD formula (ng/ml) RT (min) R 2 Intraday %RSD Interday % RSD ATP C 10 H 15 N 5 O 13 P 3 dgtp C 10 H 15 N 5 O 13 P 3 ITP C 10 H 14 N 4 O 14 P 3 GTP C 10 H 15 N 5 O 14 P 3 UDPDglucose C 15 H 23 N 2 O 17 P 2 UDPDglucuronate C 15 H 21 N 2 O 18 P 2 ADPDglucose C 16 H 24 N 5 O 15 P 2 Guanosine 5'diphosphate,3' diphosphate C 10 H 16 N 5 O 17 P 4 UDPNacetylDglucosamine C 17 H 26 N 3 O 17 P 2 Oxidized glutathione C 20 H 33 N 6 O 12 S 2 NAD C 21 H 26 N 7 O 14 P 2 NADH C 21 H 28 N 7 O 14 P 2 506 516 21 408 418 C 10 H 12 N 5 O 9 P 2 506 516 28 159 159 HO 6 P 2 507 517 19 409 419 C 10 H 11 N 4 O 10 P 2 522 532 23 424 434 C 10 H 12 N 5 O 10 P 2 500 28.9 0.999 2 15 100 32.8 0.95 6 11 100 32.1 0.998 16 14 500 29.5 0.922 b 6 b 30 b 565 580 23 323 332 C 9 H 12 N 2 O 9 P 100 24.5 0.999 22 12 579 594 24 403 4 9 H 13 N 2 O 12 P 2 25 25.2 0.965 25 20 588 604 22 346 356 C 10 H 13 N 5 O 7 P 50 24.7 0.982 12 6 602 612 22 504 514 C 10 H 13 N 5 O 13 P 3 606 623 26 385 394 C 9 H 11 N 2 O 11 P 2 613 633 33 231 239 C 8 H 11 N 2 O 2 S 2 662 683 19 540 555 C 15 H 20 N 5 O 13 P 2 664 685 31 408 418 C 10 H 12 N 5 O 9 P 2 1500 46 n/a n/a n/a 100 24.5 0.948 20 24 50 24.8 0.991 10 24 5 16.5 0.997 16 17 a 500 25.2 0.923 43 54 DephosphoCoA C 21 H 36 N 7 O 13 P 2 S 688 709 25 348 358 C 10 H 15 N 5 O 7 P 50 25 0.989 17 21 NADP C 21 H 27 N 7 O 17 P 3 742 763 18 620 635 C 15 H 20 N 5 O 16 P 3 50 25 0.997 12 10 NAD C 21 H 29 N 7 O 17 P 3 744 765 34 408 418 C 10 H 12 N 5 O 9 P 2 1500 26.3 n/a n/a n/a CoA C 21 H 37 N 7 O 16 P 3 S 768 789 37 261 272 C 11 H 21 N 2 O 3 S 50 26.4 0.994 17 21 FAD C 27 H 34 N 9 O 15 P 2 786 813 24 348 358 C 10 H 15 N 5 O 7 P 10 25 0.99 7 20 AcetylCoA C 23 H 39 N 7 O 17 P 3 S 810 833 28 303 316 C 13 H 23 N 2 O 4 S 250 26 0.970 b 2 b 13 b PropionylCoA C 24 H 41 N 7 O 17 P 3 S 824 848 33 317 331 C 14 H 25 N 2 O 4 S 25 26 0.985 9 14 AcetoacetylCoA C 25 H 41 N 7 O 18 P 3 S 852 877 34 345 360 C 15 H 15 N 2 O 5 S 50 28 0.985 b 6 b 17 b MalonylCoA C 24 H 39 N 7 O 19 P 3 S 854 878 26 347 361 C 14 H 23 N 2 O 6 S a 100 27.3 0.993 11 11 SuccinylCoA C 25 H 41 N 7 O 19 P 3 S 868 893 38 361 376 C 15 H 25 N 2 O 6 S 25 27.2 0.967 7 11 3Hydroxy3 methylglutarylcoa C 27 H 43 N 7 O 20 P 3 S 910 937 43 408 418 C 10 H 12 N 5 O 9 P 2 500 27.1 0.983 24 18 Bajad et al., 2006 Supplementary Materials Page S10 of 17

* Charge on the ion indicates ionization mode used for the metabolite The product ion formula is confirmed only for compounds found in Supplementary Table 4; for other compounds, the product ion and formula are best guesses based on available literature Stable ( 20% loss) when stored for 1 week at 4 C at ph 2.8 a Unstable (> 20% loss) when stored for 1 week at 4 C at ph 2.8, but stable ( 20% loss) when stored for 1 week at 4 C at ph 6.8 Unstable (> 20% loss) when stored for 1 week at 4 C at ph 2.8 or ph 6.8. Note that, except for oxaloaceate (at ph 6.8), erythrose4 phosphate (at ph 2.8 and 6.8), deoxyribosephosphate (at ph 2.8), and PRPP (at ph 2.8), t 1/2 for all compounds is > 24 hrs at 4 C Linearity is not reported for unstable metabolites or metabolites with LOD 1000 ng/ml b Reported results are for peak area, not height Intra and interday assay variation is not reported for unstable metabolites or metabolites with LOD > 1000 ng/ml n/a not applicable Bajad et al., 2006 Supplementary Materials Page S11 of 17

Table S3: Performance of different chromatography conditions for distinct cellular metabolites. The chromatography columns and conditions employed are described in Table 2 of the main text. The numerical values found in here refer to the overall performance score for the specified metabolite under the stated chromatography conditions as determined in the Methods, with values > 500 considered good, 250 500 fair, and < 250 poor. Metabolite name ion formula Reverse Phase HILIC ph 2.5 ph 5.8 ph 9 ph 2.8 ph 5.8 ph 9.0 ion 76 100 144 131 119 141 43 112 355 62 0 68 659 601 222 398 185 112 250 89 239 75 161 458 202 0 0 0 0 0 0 909 691 380 633 0 0 0 90 181 202 87 262 206 149 118 124 109 64 98 459 720 240 601 266 224 693 101 Unstable Bajad et al., 2006 Supplementary Materials Page S12 of 17 Glycine C 2 H 6 NO 2 Putrescine C 4 H 13 N 2 Alanine C 3 H 8 NO 2 Acetoacetate C 4 H 5 O 3 Choline C 5 H 14 NO 104 378 275 284 455 0 225 243 1019 548 589 414 764 285 485 443 366 425 543 Glycerate C 3 H 5 O 4 105 0 0 0 0 0 0 0 0 0 0 0 20 0 56 285 119 75 677 Serine C 3 H 8 NO 3 106 180 256 232 102 240 185 204 157 167 125 163 839 871 497 486 246 298 569 Cytosine C 4 H 6 N 3 O 112 166 247 534 380 223 198 250 178 84 285 138 731 823 186 792 423 531 310 Uracil C 4 H 5 N 2 O 2 113 164 0 349 174 229 0 0 70 0 212 117 215 220 30 350 26 0 0 Fumarate C 4 H 3 O 4 115 0 297 99 90 0 0 42 27 26 194 0 118 0 92 0 172 68 0 Proline C 5 H 10 NO 2 116 405 298 245 261 268 395 206 165 494 122 308 829 902 516 875 498 226 1015 Succinate C 4 H 5 O 4 117 0 0 0 0 0 0 107 50 107 247 109 189 188 0 99 0 0 128 Threonine C 4 H 8 NO 3 118 173 224 168 106 130 64 156 122 154 78 93 280 435 295 484 127 61 354 Valine C 5 H 12 NO 2 118 441 243 202 426 107 182 166 183 320 38 148 669 718 280 702 535 244 952 Homoserine C 4 H 10 NO 3 120 115 139 158 72 127 0 0 0 0 100 0 557 409 303 396 438 0 0 Cysteine C 3 H 8 NO 2 S 122 116 315 274 109 146 14 95 46 118 52 91 402 92 529 465 308 204 134 Nicotinate C 6 H 4 NO 2 122 48 379 582 185 192 173 284 158 125 594 224 650 609 268 635 252 419 405 Nicotinamide C 6 H 7 N 2 O 123 79 624 837 420 270 626 98 501 542 639 253 739 538 184 752 565 669 345 Taurine C 2 H 6 NO 3 S 124 70 78 174 110 99 56 83 75 0 149 0 123 641 196 605 161 420 640 Thymine C 5 H 7 N 2 O 2 127 0 403 395 289 380 0 210 74 163 0 117 199 151 263 391 0 80 286 Agmatine C 5 H 15 N 4 131 216 271 128 348 220 0 0 0 0 94 0 700 683 451 589 0 0 199 Oxaloacetate C 4 H 3 O 5 131 Unstable Aspartate C 4 H 6 NO 4 132 185 208 63 128 101 191 149 135 106 192 201 494 189 121 224 226 195 460 (Iso)leucine C 6 H 14 NO 2 132 166 566 244 359 336 322 361 471 458 182 193 855 984 1192 869 385 462 1129 Asparagine C 4 H 9 N 2 O 3 133 213 259 420 220 225 179 81 38 83 0 27 739 801 1023 554 474 40 463

Metabolite name ion formula Reverse Phase HILIC ph 2.5 ph 5.8 ph 9 ph 2.8 ph 5.8 ph 9.0 ion 133 220 363 201 358 96 129 224 57 78 232 140 182 0 49 102 0 0 1069 133 184 284 255 254 330 140 300 499 687 0 381 462 819 559 677 375 177 766 136 483 394 0 623 286 254 597 594 857 0 336 755 580 523 893 308 641 1149 Malate C 4 H 5 O 5 Ornithine C 5 H 13 N 2 O 2 Adenine C 5 H 6 N 5 Homocysteine C 4 H 10 NO 2 S 136 Added after chromatography optimization paminobenzoate/ C 7 H 6 NO 2 136 556 841 0 659 1036 0 213 68 131 182 34 131 512 240 474 0 200 0 Anthranilate Hypoxanthine C 5 H 5 N 4 O 137 143 645 477 373 54 367 265 148 218 138 239 391 610 107 827 136 413 660 phydroxybenzoate C 7 H 5 O 3 137 598 604 0 518 461 87 28 138 108 657 155 13 0 0 0 412 168 481 Acetylphosphate C 2 H 4 O 5 P Unstable Carbamoylphosphate CH 3 NO 5 P 140 Added after chromatography optimization and unstable Histidinol C 6 H 12 N 3 O 142 221 266 104 528 263 0 162 815 309 0 188 564 563 603 797 522 0 1018 αketoglutarate C 5 H 5 O 5 145 0 125 92 77 0 66 101 131 80 0 89 66 0 73 29 76 42 205 Spermidine C 7 H 20 N 3 146 284 281 0 381 209 0 0 0 0 0 0 337 270 152 233 0 0 0 Glutamine C 5 H 11 N 2 O 3 147 324 437 433 404 332 584 319 207 434 516 238 1051 1089 820 874 659 628 1014 Lysine C 6 H 15 N 2 O 2 147 271 344 291 456 322 131 210 604 402 0 237 650 809 583 748 469 189 611 Glutamate C 5 H 10 NO 4 148 267 271 74 210 1285 199 261 239 339 344 294 556 764 101 648 522 412 600 Methionine C 5 H 12 NO 2 S 150 151 361 344 273 249 292 145 333 262 97 179 833 512 312 937 327 435 833 Guanine C 5 H 6 N 5 O 152 131 0 0 133 165 0 133 98 182 0 0 240 308 38 174 166 138 494 2,3Dihydroxybenzoate C 7 H 5 O 4 153 0 951 84 450 0 317 190 19 194 0 0 160 0 123 269 169 307 729 Xanthine C 5 H 5 N 4 O 2 153 0 575 0 366 361 0 153 70 336 0 0 86 783 80 724 0 157 0 Orotate C 5 H 3 N 2 O 4 155 453 256 25 221 0 206 306 295 195 0 372 85 0 102 865 528 295 594 Histidine C 6 H 10 N 3 O 2 156 205 290 252 227 268 63 137 478 330 0 505 451 941 859 686 88 0 492 Dihydroorotate C 5 H 5 N 2 O 4 157 163 241 634 303 0 103 157 157 150 626 245 68 0 177 710 158 310 589 Allantoin C 4 H 7 N 4 O 3 159 134 153 423 31 216 74 97 93 0 0 0 153 348 0 0 0 0 0 Carnitine C 7 H 16 NO 3 162 439 366 333 482 303 266 285 264 316 167 189 998 773 389 974 871 784 1431 Phenylpyruvate C 9 H 7 O 3 163 0 733 0 325 0 0 388 182 1101 960 128 166 0 262 125 149 86 200 Phenylalanine C 9 H 12 NO 2 166 493 948 1255 589 332 638 472 240 941 326 286 714 870 329 873 260 476 1420 Quinolinate C 7 H 4 NO 4 166 0 104 0 0 0 88 182 76 210 0 315 19 0 53 0 123 0 722 Phosphoenolpyruvate C 3 H 4 O 6 P 167 159 28 0 40 0 215 197 111 112 173 333 60 0 0 0 191 91 515 Bajad et al., 2006 Supplementary Materials Page S13 of 17

Metabolite name ion formula ion Reverse Phase HILIC ph 2.5 ph 5.8 ph 9 ph 2.8 ph 5.8 ph 9.0 Dihydroxyacetonephosphate C 3 H 6 O 6 P 169 Added after chromatography optimization Pyridoxine C 8 H 12 NO 3 170 196 944 989 595 331 757 474 39 1036 656 820 451 770 172 1073 433 107 1924 Glycerol3phosphate C 3 H 8 O 6 P 171 Added after chromatography optimization Arginine C 6 H 13 N 4 O 2 173 239 270 222 403 330 77 373 646 353 0 87 568 730 619 602 463 82 572 Shikimate C 7 H 9 O 5 173 186 69 785 70 743 48 92 87 99 217 164 90 217 90 688 412 189 564 Aconitate C 6 H 5 O 6 173 95 799 152 214 360 170 264 164 194 260 433 190 0 175 406 212 92 631 Citrulline C 6 H 12 N 3 O 3 174 131 161 160 215 282 591 225 248 257 167 83 956 955 757 672 708 648 724 Allantoate C 4 H 7 N 4 O 4 175 477 329 1026 258 0 152 137 209 204 98 402 92 0 126 760 479 227 396 CarbamoylLaspartate C 5 H 7 N 2 O 5 175 Added after chromatography optimization NαAcetylornithine C 7 H 15 N 2 O 3 175 211 288 251 369 270 281 256 141 268 242 147 858 457 783 794 755 735 537 Hydroxyphenylpyruvate C 9 H 7 O 4 179 0 326 104 0 88 167 296 57 706 0 0 79 0 204 0 208 88 135 myoinositol C 6 H 11 O 6 179 0 0 0 0 0 0 0 0 0 0 0 0 83 0 0 0 0 0 Glucosamine C 6 H 14 NO 5 180 270 251 190 292 246 376 260 524 315 104 208 838 697 123 758 86 404 916 Homocysteic acid C 4 H 8 NO 5 S 182 369 141 410 158 0 95 143 161 115 292 147 222 0 169 646 145 523 408 Tyrosine C 9 H 12 NO 3 182 93 490 0 372 486 456 263 431 382 0 185 860 685 244 549 348 246 987 3Phosphoglycerate C 3 H 6 O 7 P 185 0 41 0 87 385 80 134 154 92 139 28 59 0 0 0 0 0 230 3Phosphoserine C 3 H 9 NO 6 P 186 215 89 115 162 0 182 119 143 202 343 399 0 0 0 0 48 0 892 Citrate C 6 H 7 O 7 191 0 246 0 99 0 229 250 143 211 319 292 323 0 0 0 0 0 0 2DehydroDgluconate C 6 H 9 O 7 193 0 154 227 62 0 202 150 86 116 0 256 115 0 0 28 54 245 684 Gluconate C 6 H 11 O 7 195 305 244 355 177 54 178 199 67 147 0 308 188 0 50 179 94 83 393 Erythrose4phosphate C 4 H 8 O 7 P 199 Unstable Tryptophan C 11 H 13 N 2 O 2 205 1052 1365 213 938 630 575 662 698 504 0 308 727 875 657 810 546 550 1220 DGlucarate C 6 H 9 O 8 209 Added after chromatography optimization Deoxyribosephosphate C 5 H 10 O 7 P 213 Unstable Pantothenate C 9 H 16 NO 5 218 1192 1041 1090 457 882 211 178 242 41 614 195 95 767 235 909 278 857 375 Cystathionine C 7 H 15 N 2 O 4 S 223 349 374 85 201 87 208 297 263 200 84 344 664 750 493 1106 313 77 741 Prephenate C 10 H 9 O 6 225 Unstable Deoxyuridine C 9 H 13 N 2 O 5 229 36 485 784 395 113 423 382 402 373 403 291 225 431 49 705 76 489 458 DRib(ul)ose5phosphate C 5 H 10 O 8 P 229 310 214 82 231 0 148 229 155 167 336 121 261 0 113 56 231 182 450 Bajad et al., 2006 Supplementary Materials Page S14 of 17

Metabolite name ion formula Reverse Phase HILIC ph 2.5 ph 5.8 ph 9 ph 2.8 ph 5.8 ph 9.0 ion 241 963 958 912 439 169 751 608 267 872 0 390 194 646 60 731 221 720 868 243 130 219 1060 461 142 145 517 494 200 456 208 760 604 271 818 359 821 981 244 309 115 1564 730 375 262 570 592 415 1000 552 921 1090 379 871 472 774 1564 Thymidine C 10 H 13 N 2 O 5 Uridine C 9 H 11 N 2 O 6 Cytidine C 9 H 14 N 3 O 5 Biotin C 10 H 17 N 2 O 3 S 245 1010 1162 1103 927 956 585 566 198 748 1041 295 81 726 138 1021 473 968 1010 Deoxyadenosine C 10 H 14 N 5 O 3 252 1107 1207 0 680 361 1000 1082 776 1108 0 429 1113 467 181 997 259 825 1588 Deoxyinosine C 10 H 13 N 4 O 4 253 459 1065 0 461 291 814 784 537 595 0 321 0 945 231 905 287 765 914 DHexosephosphate C 6 H 12 O 9 P 259 292 186 203 278 0 186 219 173 152 323 266 216 97 91 85 218 236 511 Glucosamine1P C 6 H 15 NO 8 P 260 250 176 75 247 839 185 246 255 199 216 407 135 717 233 274 196 50 770 Glucosamine6P C 6 H 15 NO 8 P 260 145 327 164 287 744 132 267 236 237 350 294 115 374 117 177 51 0 724 Thiamine C 12 H 17 N 4 OS 265 129 313 720 555 295 824 1831 1560 997 0 135 973 769 931 957 226 110 1160 Adenosine C 10 H 14 N 5 O 4 268 Added after chromatography optimization Deoxyguanosine C 10 H 14 N 5 O 4 268 888 1552 0 586 678 1075 865 474 1015 0 509 874 1022 491 939 132 772 1332 Inosine C 10 H 13 N 4 O 5 269 705 850 0 487 643 763 613 418 614 0 294 0 960 212 923 290 983 987 6PhosphoDgluconate C 6 H 12 O 10 P 275 27 147 83 177 0 87 51 81 41 83 57 146 0 66 0 0 0 264 Guanosine C 10 H 14 N 5 O 5 284 706 1082 0 393 552 599 414 367 434 0 242 635 953 265 971 209 783 1085 Xanthosine C 10 H 13 N 4 O 6 285 868 893 0 412 958 285 285 238 513 305 259 0 859 200 1587 174 807 617 NAcetylglucosamine1 C 8 H 15 NO 9 P 300 310 220 241 231 0 146 152 149 140 111 171 220 0 363 191 354 126 526 phosphate dump C 9 H 12 N 2 O 8 P 307 104 43 432 237 0 146 215 192 99 731 236 101 0 81 0 171 196 504 dcmp C 9 H 15 N 3 O 7 P 308 103 89 0 219 0 274 307 194 283 803 418 322 0 528 194 289 434 817 Reduced glutathione C 10 H 18 N 3 O 6 S 308 78 151 0 543 0 70 200 108 154 0 32 711 0 512 523 541 137 234 GeranylPP C 10 H 19 O 7 P 2 313 0 436 33 0 0 623 320 53 630 0 361 0 0 0 0 90 148 382 dtmp C 10 H 16 N 2 O 8 P 323 657 69 55 239 0 162 209 267 457 0 263 201 0 220 57 284 414 653 CMP C 9 H 15 N 3 O 8 P 324 203 397 0 307 0 255 172 238 221 874 205 282 0 289 240 196 222 794 UMP C 9 H 14 N 2 O 9 P 325 416 188 0 160 0 349 197 176 122 758 226 211 0 56 0 174 343 581 cyclicamp C 10 H 11 N 5 O 6 P 328 1024 314 0 439 67 271 831 293 794 0 154 413 0 749 787 134 1020 495 damp C 10 H 15 N 5 O 6 P 332 107 174 97 225 38 257 259 207 352 0 312 292 0 254 70 410 249 747 Fructose1,6bisphosphate C 6 H 13 O 12 P 2 339 Added after chromatography optimization Trehalose C 12 H 21 O 11 341 0 59 50 0 81 0 0 0 0 0 0 0 376 0 0 0 150 307 Thiaminephosphate C 12 H 18 N 4 O 4 PS 345 484 353 806 655 184 331 172 157 602 0 493 331 596 504 288 233 0 974 Bajad et al., 2006 Supplementary Materials Page S15 of 17

Metabolite name ion formula ion Reverse Phase HILIC ph 2.5 ph 5.8 ph 9 ph 2.8 ph 5.8 ph 9.0 AMP C 10 H 15 N 5 O 7 P 348 156 73 0 225 102 249 98 233 431 0 304 276 0 100 75 230 187 874 dgmp C 10 H 15 N 5 O 7 P 348 506 293 259 114 0 104 113 152 314 0 255 89 0 0 0 86 135 647 IMP C 10 H 14 N 4 O 8 P 349 4 45 0 193 38 55 101 271 274 0 476 221 0 47 0 214 218 758 GMP C 10 H 15 N 5 O 8 P 364 251 276 0 218 0 167 263 142 291 0 637 94 0 0 0 193 202 1203 Xanthosine5phosphate C 10 H 14 N 4 O 9 P 365 407 202 0 215 0 120 140 200 139 318 330 100 0 15 0 46 54 603 Orotidinephosphate C 10 H 12 N 2 O 11 P 367 Added after chromatography optimization Riboflavin C 17 H 21 N 4 O 6 377 901 1241 0 867 452 854 1071 1225 1140 0 477 142 976 0 893 284 989 1598 trans, transfarnesyl diphosphate C 15 H 27 O 7 P 2 381 0 0 0 0 0 251 0 494 0 0 0 0 51 0 0 107 193 0 SAdenosylLhomocysteine C 14 H 21 N 6 O 5 S 385 743 615 0 567 298 1019 705 424 996 0 395 740 788 696 754 549 503 587 dcdp C 9 H 14 N 3 O 10 P 2 386 0 60 0 87 0 130 145 102 94 206 335 21 0 0 0 186 0 275 PRPP C 5 H 12 O 14 P 3 389 0 0 0 0 0 181 103 83 122 189 83 67 0 0 0 0 0 251 SAdenosylLmethionine C 15 H 23 N 6 O 5 S 399 205 404 0 498 262 0 468 681 1043 0 362 512 619 565 650 172 0 878 dtdp C 10 H 15 N 2 O 11 P 2 401 0 0 0 0 0 185 131 101 233 0 223 23 0 0 0 113 65 481 CDP C 9 H 14 N 3 O 11 P 2 402 0 32 0 96 0 122 112 109 49 310 137 34 0 0 0 67 118 344 UDP C 9 H 13 N 2 O 12 P 2 403 0 14 0 49 0 13 43 0 62 0 0 19 0 0 0 0 5 0 ADP C 10 H 14 N 5 O 10 P 2 426 42 123 0 92 0 175 135 92 198 0 251 29 0 38 117 209 166 279 APS C 10 H 13 N 5 O 10 PS 426 580 294 0 315 0 361 308 188 410 0 899 146 0 89 294 304 757 1208 dgdp C 10 H 14 N 5 O 10 P 2 426 54 110 0 66 0 180 181 118 270 0 122 27 0 50 211 282 83 385 IDP C 10 H 13 N 4 O 11 P 2 427 85 0 0 40 0 70 40 42 0 0 0 0 0 0 0 145 0 0 Folate C 19 H 20 N 7 O 6 442 731 1171 0 423 0 630 471 92 0 0 0 591 0 0 677 492 318 598 GDP C 10 H 14 N 5 O 11 P 2 442 0 0 0 0 0 109 83 169 78 0 34 0 0 0 0 0 0 198 7,8Dihydrofolate C 19 H 22 N 7 O 6 444 Unstable FMN C 17 H 20 N 4 O 9 P 455 543 721 0 500 0 640 1000 132 880 0 128 129 66 0 0 190 135 416 5MethylTHF C 20 H 26 N 7 O 6 460 1020 1507 0 60 92 618 957 294 665 573 545 644 0 329 0 531 110 758 dctp C 9 H 15 N 3 O 13 P 3 466 0 0 0 81 0 132 127 108 98 0 326 0 0 0 0 0 58 308 dutp C 9 H 14 N 2 O 14 P 3 467 0 0 0 31 0 132 136 107 96 0 10 0 0 0 0 27 0 310 TTP C 10 H 16 N 2 O 14 P 3 481 0 0 0 0 0 141 123 105 110 0 8 0 0 0 0 116 40 367 CTP C 9 H 15 N 3 O 14 P 3 482 0 0 0 55 0 128 142 122 122 0 91 0 0 0 0 0 65 390 Bajad et al., 2006 Supplementary Materials Page S16 of 17

Metabolite name ion formula Reverse Phase HILIC ph 2.5 ph 5.8 ph 9 ph 2.8 ph 5.8 ph 9.0 ion 483 0 0 0 0 0 107 131 105 84 0 0 0 0 0 0 45 0 270 490 0 0 0 82 0 183 165 117 249 0 0 0 0 0 0 43 0 509 506 0 0 0 0 0 139 125 93 306 0 0 0 0 0 0 58 0 246 506 0 0 0 0 0 138 156 113 324 0 0 0 0 0 0 35 0 279 507 0 0 0 0 0 147 128 89 101 0 0 0 0 0 0 0 0 255 522 0 0 0 0 0 110 112 97 71 0 0 0 0 0 0 0 0 126 565 181 127 0 247 0 213 210 204 171 601 339 116 0 51 93 287 501 649 579 Added after chromatography optimization 588 364 296 0 265 0 234 191 147 188 0 295 140 0 84 179 188 426 586 602 Added after chromatography optimization and unstable UTP C 9 H 14 N 2 O 15 P 3 datp C 10 H 15 N 5 O 12 P 3 ATP C 10 H 15 N 5 O 13 P 3 dgtp C 10 H 15 N 5 O 13 P 3 ITP C 10 H 14 N 4 O 14 P 3 GTP C 10 H 15 N 5 O 14 P 3 UDPDglucose C 15 H 23 N 2 O 17 P 2 UDPDglucuronate C 15 H 21 N 2 O 18 P 2 ADPDglucose C 16 H 24 N 5 O 15 P 2 Guanosine 5' C 10 H 16 N 5 O 17 P 4 diphosphate,3'diphosphate UDPNacetylDglucosamine C 17 H 26 N 3 O 17 P 2 606 38 118 0 181 0 189 159 97 145 353 214 115 0 83 71 282 297 437 Oxidized glutathione C 20 H 33 N 6 O 12 S 2 613 395 194 0 397 0 0 160 217 166 131 351 311 0 0 0 24 37 457 NAD C 21 H 26 N 7 O 14 P 2 662 194 122 0 236 0 111 402 224 676 0 416 267 0 231 312 323 508 591 NADH C 21 H 28 N 7 O 14 P 2 664 73 58 0 126 0 152 0 102 472 0 174 117 0 59 71 363 676 509 DephosphoCoA C 21 H 36 N 7 O 13 P 2 S 688 58 751 0 101 0 315 0 53 316 0 0 42 0 0 0 304 484 71 NADP C 21 H 27 N 7 O 17 P 3 742 34 58 0 157 0 111 56 189 491 0 394 90 0 0 0 0 0 643 NAD C 21 H 29 N 7 O 17 P 3 744 Unstable CoA C 21 H 37 N 7 O 16 P 3 S 768 0 0 0 0 0 259 0 70 0 0 0 0 0 0 0 0 0 56 FAD C 27 H 34 N 9 O 15 P 2 786 0 137 0 739 0 937 993 437 953 0 369 176 0 0 0 664 544 843 AcetylCoA C 23 H 39 N 7 O 17 P 3 S 810 0 0 0 0 0 302 0 29 0 0 0 0 0 0 0 0 0 250 PropionylCoA C 24 H 41 N 7 O 17 P 3 S 824 Added after chromatography optimization AcetoacetylCoA C 25 H 41 N 7 O 18 P 3 S 852 0 101 0 0 0 288 0 41 0 0 0 68 0 0 0 0 0 260 MalonylCoA C 24 H 39 N 7 O 19 P 3 S 854 0 0 0 0 0 34 43 91 0 0 0 0 0 0 0 0 0 587 SuccinylCoA C 25 H 41 N 7 O 19 P 3 S 868 Added after chromatography optimization 3Hydroxy3 methylglutarylcoa C 27 H 43 N 7 O 20 P 3 S 910 Added after chromatography optimization Bajad et al., 2006 Supplementary Materials Page S17 of 17