Supplementary Information For:

Development and Evaluation of a Parallel Reaction Monitoring Strategy for Large-Scale of Targeted

Metabolomics Quantification

Juntuo Zhou1, Huiying Liu1, Yang Liu, Jia Liu, Xuyang Zhao, Yuxin Yin*

Supplementary Methods

Materials.

RPMI 1640 medium was purchased from Cellgro. Fetal Bovine Serum (FBS), penicillin, and streptomycin were purchased from

Hyclone Laboratories. Ammonium acetate was purchased from Sigma. HPLC grade ammonium hydroxide, acetonitrile and methanol

were purchased from Fisher Scientific. Deionized water was produced by a Milli-Q system. Sample Preparation

Cell culture.

The human cell lines HEK293T and, HCT116, and mouse embryonic fibroblasts (MEFs) were purchased from the American Type

Culture Collection (ATCC, USA). Cells were grown in RPMI 1640 with 10 % FBS, 100 units/mL penicillin and 100 μg/mL

streptomycin at 37 °C in a humidified incubator with 5 % CO2.

Serum specimens from T2DM patients and healthy controls.

Primary serum specimens from 19 T2DM patients or healthy people were obtained from China-Japan Friendship Hospital. The study

was approved by the local ethics committee for clinical studies. Only samples from patients or healthy people who agreed to

participate for the purpose of laboratory research were used.

Sample preparation for concentration curves study.

The metabolites used for concentration curves were from the serum sample. 200 μL of serum sample was mixed with a 4-fold volumes

of cold methanol, vortexed for 5 min and centrifuged at 14000 g for 15 min at 4 °C. The supernatant was then transferred to new

eppendorf tubes and dried in a SpeedVac. After drying, each sample was reconstituted into 100 μL of deionized water. A serial

dilution was performed, representing the amount of metabolites extracted from 100, 20, 4, 0.8, 0.16 and 0.032 μL of serum samples.

Concentration curves in triplicate were acquired on a Q-Exactive instrument in PRM mode.

Preparation of cells, medium and serum for metabolic determination.

Three kinds of cells were grown in three 10 cm dishes with full growth medium. When cells reached 80 % confluence, 200 μL of the

media was collected and mixed with 800 μL of methanol and kept at -80 °C for 20 min. Cells in dishes were washed with 0.9 % saline

and 1 mL of extraction solvent (methanol/water 4:1 v/v) was immediately added. Cells were then stored at −80 °C for 20 min and

scraped into EP tubes. Medium and cells were oscillated for 10 min and centrifuged at 14000 g for 10 min at 4 °C. Serum (human and

mouse) samples were mixed with 4-fold volumes of cold methanol, vortexed for 5 min and centrifuged at 14000 g for 15 min at 4 °C.

Supernatant from media, cells or serum was then transferred to new eppendorf tubes and dried in a SpeedVac. After drying, all

samples were stored at −80 °C before use. For the LC-MS assay, samples were reconstituted into 100 μL of water (LC-MS grade,

Fisher Scientific), and 5 μL was used for analysis be LC-MS/MS.

For the T2DM serum samples, serum was mixed with a 4-fold volumes of cold methanol, vortexed for 5 min and centrifuged at 14000

g for 15 min at 4 °C. The supernatant was then transferred to new eppendorf tubes and dried in a SpeedVac. For the LC-MS assay,

samples were reconstituted into 100 μL of water (LC-MS grade, Fisher Scientific), and 5 μL was injected into the LC-MS/MS. A QC

sample consisted of an equal mixture of all 38 samples, and this was analyzed repeatedly during the period of whole T2DM sample

analysis (one analysis at the beginning, two in the middle and one at the end) using the same method as the T2DM samples to monitor

the stability of LC-MS performance.

High-performance liquid chromatography.

For the PRM assay, the Ultimate 3000 UHPLC system was coupled to Q-Exactive MS (Thermo Scientific) for metabolite separation

and detection. An Xbridge amide column (100 × 4.6 mm i.d., 3.5 μm; Waters) was employed for compound separation at 30 °C. The

mobile phase A was 5 mM ammonium acetate in water with 5 % acetonitrile, and mobile phase B was acetonitrile. The linear gradient

used was as follows: 0 min, 90 % B; 3 min, 30 % B; 12 min, 2 % B; 15 min, 2 % B, 16 min, 90 % B and 23 min, 90 % B. The flow

rate was 0.5 mL/min. For the MRM assay, the Nexera UHPLC system (Shimazu) was coupled to QTRAP 6500 MS (AB Sciex) using

the same chromatographic conditions as above.

Mass spectrometry.

Q-Exactive – targeted metabolite MS/MS acquisition and subsequent PRM assays.

Samples were analyzed using a quadrupole Orbitrap (Q-Exactive) mass spectrometer. Typically, mass resolution for MS1 scans and

targeted metabolites MS/MS scans was 17,500. Initially, data acquisition was performed in seven unscheduled target MS/MS assay to

obtain MS/MS spectra and retention time information for over 237 candidate metabolites. The final PRM acquisition, monitoring 237

metabolites on schedule, consisted of 1 MS1 scan (53 msec) followed by targeted MS/MS scans in HCD (high-energy collisional

dissociation) mode with cycle times ranging between 1 and 6 seconds depending on target numbers. Acquisition was performed in

positive ion mode (109 metabolites) and negative ion mode (128 metabolites) using scheduled mode. Scheduled means that one

metabolite was only monitored by MS in a period around its elution time. For PRM assay, we used a monitor window of 4 minutes.

For example, the elution time of lysine is about 17.5 min, so the mass spectrum monitors the ion pair of lysine 147/89 from 15.5 min

to 19.5 min. Scheduled assay can improve the amount of metabolites measured in a single assay because it can reduce the cycle time

of the MS assay. And in MRM assay, we didn’t employ the schedule strategy because the scan speed of triple quadrupole instrument

is fast enough to deal with all metabolites in the whole assay.

QTRAP 6500 –high throughput MRM assays.

MRM analysis was performed using the QTRAP 6500, a hybrid triple quadrupole/linear ion trap (AB SCIEX). Confirmed metabolite

data (precursor-product ion pairs and retention time) obtained from PRM results were transferred to a transition list for MRM assay

(237 Q1/Q3 transitions for 237 metabolites). The MRM assay was performed in a positive-negative ion switching mode without

scheduling.

Peak extraction with Skyline.

Application of Skyline for PRM data processing was done in three steps. Step 1: the precursor ion/product ion pairs of the 237

metabolites were generated manually based on the acquired MS/MS spectra of each metabolite acquired in the metabolites

identification phase (See Figure 1A, flowchart). The product ion was determined as the one with highest intensity or with specific

character. For example, the family of CoA shares a common product ion 184.07. Then the 237 ion pairs representing the 237

metabolites and the exact retention time information were imported into Skyline. Step 2: after PRM data acquisition by LC-MS/MS,

the raw data files were imported into Skyline to perform peak extraction and peak area calculation for each ion pair that we

constructed in step 1. Step 3: peaks in the raw data were aligned to the previously imported information and confirmed as homologous

compound. Then the information of the detected metabolites including metabolite name, sample name, peak area, measured retention

time and signal to noise was exported as table format for further statistical analysis.

Figure S1

Method names

Number of metabolites Sample names

Number of metabolites

MRM 217 Serum 196

MS1 203 Medium 180

PRM 230 Cell 216

Overall number of unique metabolites 237 Overall number of unique metabolites 230

Figure S1. Venn diagram of the numbers of metabolites identified by different assay methods (Left), and in different samples

(Right, identified by PRM assay).

Figure S2

Figure S1. Cross validation (Left, method: LOOCV) and permutation test results (Right, method: Prediction accuracy during

training, permutation numbers: 2000) from the PLS-DA model with T2DM samples.

Table S1. Metabolites in the constructed PRM assay method.

m/z Formula [M] Polarity NCE Metabolite name RT(min) Q1 Q3

61.0402 CH4N2O Positive 21 Urea 5.39 61.04 61.04036

62.0606 C2H7NO Positive 53 Ethanolamine 13.35 62.06 62.06078

76.03983 C2H5NO2 Positive 20 Glycine 12.29 76.04 58.0659

89.10785 C4H12N2 Positive 20 Putrescine 14.8 89.1 72.08132

90.05548 C3H7NO2 Positive 20 Alanine 12.13 90.06 90.05535

90.0555 C3H7NO2 Positive 20 Sarcosine 12.5 90.06 72.04501

102.0919 C5H12NO Positive 20 Betaine aldehyde 10.04 103.1 95.04949

103.0395 C4H6O3 Positive 20 Acetoacetic acid 12.25 103.0395 95.04945

103.0997 C5H13NO Positive 21 Dimethylglycine 9.75 104.1072 60.08149

104.0711 C4H9NO2 Positive 20 Choline 11.7 104.07 58.06586

106.0504 C3H7NO3 Positive 20 Serine 12.66 106.05 60.0451

112.0511 C4H5N3O Positive 20 Cytosine 8.7 112.05 70.06575

113.0351 C4H4N2O2 Positive 22 Uracil 13.08 113.0351 70.06577

114.0667 C4H7N3O Positive 20 Creatinine 7.78 114.07 86.07165

116.0711 C5H9NO2 Positive 20 Proline 11.03 116.07 70.06568

118.0656 C8H7N Positive 20 Indole 10 118.07 91.05459

118.0868 C5H11NO2 Positive 20 Valine 10.82 118.09 72.08135

118.0868 C5H11NO2 Positive 21 Betaine 10.43 118.09 58.06578

120.066 C4H9NO3 Positive 20 Threonine 12.2 120.0656 74.06061

120.066 C4H9NO3 Positive 32 Homoserine 6.96 120.07 103.0544

121.0514 C5H4N4 Positive 35 Purine 10 121.05 94.07366

122.0275 C3H7NO2S Positive 20 Cysteine 13.24 122.03 76.0646

123.0558 C6H6N2O Positive 22 Nicotinamide 12.68 123.06 80.04996

127.0507 C6H8N2O2 Positive 20 Imidazoleacetic acid 17.54 127.05 81.04526

127.0507 C5H6N2O2 Positive 20 Thymine 11.31 127.05 85.07646

130.0868 C6H11NO2 Positive 20 DL-Pipecolic acid 11.13 130.09 84.08116

131.1184 C6H14N2O Positive 20 N-Acetylputrescine 12.43 131.11 114.091

132.066 C5H9NO3 Positive 20 Hydroxyproline 11.77 132.066 68.05009

132.0773 C4H9N3O2 Positive 20 Creatine 12.03 132.08 90.0553

132.1024 C6H13NO2 Positive 20 Leucine 9.88 132.1 86.09682

132.1024 C6H13NO2 Positive 20 Isoleucine 9.88 132.1 86.09682

133.0613 C4H8N2O3 Positive 20 Asparagine 12.76 133.06 74.02415

133.0977 C5H12N2O2 Positive 20 Ornithine 17.37 133.0969 70.0657

134.0453 C4H7NO4 Positive 20 Aspartate 12.24 134.0446 58.06585

136.0432 C4H9NO2S Positive 20 Methylcysteine 7.37 136.04 119.0491

137.0715 C7H8N2O Positive 27 2-methylnicotinamide 4.29 137.07 94.07328

140.9953 C2H5O5P Positive 24 Acetylphosphate 11.14 140.9953 123.0552

142.098 C6H11N3O Positive 20 Histidinol 11.11 142.1 124.0584

146.1657 C7H19N3 Positive 20 Spermidine 12.88 146.17 87.04449

147.0769 C5H10N2O3 Positive 20 Glutamine 12.51 147.08 130.0496

147.1133 C6H14N2O2 Positive 32 Lysine 17.45 147.11 130.0497

148.061 C5H9NO4 Positive 20 Glutamate 12.05 148.06 84.04484

148.061 C5H9NO4 Positive 20 O-acetyl-L-serine 9.99 148.06 106.05

150.0588 C5H11NO2S Positive 20 L-methionine 10.38 150.06 104.053

152.0572 C5H5N5O Positive 20 Guanine 30.38 152.06 135.0273

156.0773 C6H9N3O2 Positive 20 Histidine 13.33 156.08 110.0714

160.1337 C8H17NO2 Positive 20 2-Aminooctanoic acid 8.97 160.13 114.1278

162.113 C7H15NO3 Positive 20 L-Carnitine 12.06 162.11 103.0392

166.0868 C9H11NO2 Positive 30 Phenylalanine 9.71 166.09 120.0808

169.0977 C8H12N2O2 Positive 20 Pyridoxamine 3 169.1 151.0476

170.0817 C8H11NO3 Positive 24 Pyridoxine 4.92 170.08 152.0704

170.0929 C7H11N3O2 Positive 20 1-Methyl-Histidine 13.39 170.09 96.06851

175.1082 C7H14N2O3 Positive 20 N-acetylornithine 17.47 175.11 116.0707

175.1195 C6H14N4O2 Positive 20 Arginine 11.31 175.12 116.0706

176.1035 C6H13N3O3 Positive 20 Citrulline 12.81 176.1 159.0759

182.0817 C9H11NO3 Positive 39 Tyrosine 10.8 182.08 136.0754

184.0738 C5H15NO4P Positive 20 Phosphorylcholine 13.81 184.072 126.0912

189.0875 C7H12N2O4 Positive 20 N-Acetylglutamine 12.08 189.09 148.06

189.1239 C8H16N2O3 Positive 20 N6-Acetyl-L-lysine 12.04 189.12 126.0913

190.0715 C7H11NO5 Positive 20 N-acetyl-glutamate 11.28 190.07 130.0497

203.1508 C8H18N4O2 Positive 24 Ng,NG-dimethyl-L-arginine 11.68 203.15 116.0706

204.1236 C9H17NO4 Positive 27 L-Acetylcarnitine 11.08 203.15 144.1016

205.0977 C11H12N2O2 Positive 20 Tryptophan 9.74 205.1 146.0597

209.0926 C10H12N2O3 Positive 25 Kynurenine 9.8 209.09 94.06553

215.032 C5H11O7P Positive 25 Deoxyribose 1-phosphate 11.22 215.03 72.08139

222.0977 C8H15NO6 Positive 23 N-Acetylgalactosamine 10.08 221.1 126.0549

223.0752 C7H14N2O4S Positive 20 Cystathionine 10.82 223.09 134.0267

223.0759 C15H10O2 Positive 29 Flavone 14.5 223.08 165.0542

231.027 C5H11O8P Positive 20 Ribose-5-phosphate 12.3 231.03 116.0706

231.027 C5H11O8P Positive 20 D-Ribulose 5-phosphate 7.51 231.03 100.076

235.1082 C12H14N2O3 Positive 22 5-Methoxytryptophan 10.41 235.11 118.0863

244.0933 C9H13N3O5 Positive 20 Cytidine 9.58 244.09 162.0493

245.096 C10H16N2O3S Positive 20 Biotin 8.06 245.1 227.0842

260.0535 C6H14NO8P Positive 20 Glucosamine 6-phosphate 12.57 260.05 144.1026

261.0375 C6H13O9P Positive 20 Glucose 6-phosphate 12.7 261.04 98.98456

265.1123 C12H16N4OS Positive 20 Thiamine 13.21 265.11 122.0712

282.1202 C11H15N5O4 Positive 27 1-Methyladenosine 12.71 282.12 150.0772

284.0995 C10H13N5O5 Positive 35 Guanosine 10.05 284.1 152.0565

291.1304 C10H18N4O6 Positive 37 Argininosuccinic acid 13.41 291.13 84.08125

298.0974 C11H15N5O3S Positive 29 5'-Methylthioadenosine 3.5 298.1 136.0617

298.1151 C11H15N5O5 Positive 24 7-methylguanosine 12.21 298.11 177.0325

308.0647 C9H14N3O7P Positive 20 dCMP 12.09 308.06 76.02203

324.0597 C9H14N3O8P Positive 20 Cytidine monophosphate 12.34 324.05 112.0502

348.0709 C10H14N5O7P Positive 23 3'-AMP 11.78 348.07 136.0615

364.0658 C10H14N5O8P Positive 21 Guanosine monophosphate 12.54 364.07 152.0563

377.1461 C17H20N4O6 Positive 26 Riboflavin 9.04 377.15 91.05464

399.145 C15H22N6O5S Positive 20 S-Adenosylmethionine 16.82 399.15 250.0928

442.1475 C19H19N7O6 Positive 30 Folate 11.85 442.15 295.0932

444.1631 C19H21N7O6 Positive 32 Dihydrofolic acid 2.29 444.16 89.0601

488.1073 C14H26N4O11P2 Positive 21 CDP-choline 2.32 488.1073 360.2089

492.0086 C10H16N5O12P3 Positive 29 dATP 12.99 492.0086 136.0616

500.3046 C26H45NO6S Positive 32 Taurodeoxycholic acid 2.45 500.3046 59.04988

508.9876 C10H15N4O14P3 Positive 25 Inosine triphosphate 13.25 508.99 136.0616

523.9985 C10H16N5O14P3 Positive 25 Guanosine triphosphate 2.95 523.9985 184.0729

663.1091 C21H28N7O14P2 Positive 41 NAD 12.68 663.12 136.0616

664.117 C21H29N7O14P2 Positive 53 NADH 12.68 664.12 136.0616

688.1567 C21H35N7O13P2S Positive 37 Dephospho-CoA 2.95 688.1567 184.0731

743.0755 C21H29N7O17P3 Positive 40 NADP+ 13.06 743.08 136.0616

744.0833 C21H30N7O17P3 Positive 56 NADPH 13.06 744.0833 136.0616

768.123 C21H36N7O16P3S Positive 20 Coenzyme A 2.96 768.123 184.073

786.1649 C27H33N9O15P2 Positive 26 FAD 11.62 786.16 439.1006

808.118 C23H38N7O17P3S Positive 53 Acetyl-CoA 2.94 808.12 184.0729

824.1492 C24H40N7O17P3S Positive 33 Propionyl-CoA 2.96 824.1492 184.073

838.1649 C25H42N7O17P3S Positive 28 Butyryl-CoA 2.96 838.1649 184.0728

852.1441 C25H40N7O18P3S Positive 32 Acetoacetyl-CoA 2.37 852.1441 184.0731

854.1234 C24H38N7O19P3S Positive 26 Malonyl-CoA 2.97 854.1234 184.0726

854.1598 C25H42N7O18P3S Positive 33 3-hydroxybutyryl-CoA 2.96 854.1598 184.0729

868.1391 C25H40N7O19P3S Positive 22 Succinyl-CoA 2.96 868.1391 184.0731

75.0082 C2H4O3 Negative 38 Glycolic acid 7.13 75 72.99132

87.00824 C3H4O3 Negative 20 Pyruvic acid 11.47 87.01 72.99139

89.0721 C3H6O3 Negative 20 L-Lactic acid 8.9 89.02 87.00702

101.0239 C4H6O3 Negative 20 2-Ketobutyric acid 11.42 101.02 73.02776

105.0188 C3H6O4 Negative 20 Glyceric acid 8.52 105.02 75.007

115.0032 C4H4O4 Negative 20 Fumaric acid 7.98 115 71.01208

115.0032 C4H4O4 Negative 20 Maleic acid 7.98 115 71.01208

115.0395 C5H8O3 Negative 20 Alpha-ketoisovaleric acid 2.01 115.04 68.99396

116.046 C3H7N3O2 Negative 24 Guanidoacetic acid 9.9 116.05 74.02299

117.0188 C4H6O4 Negative 20 Methylmalonic acid 10.49 117.02 73.02776

117.0188 C4H6O4 Negative 20 Succinate 6.73 117.02 73.02771

122.0242 C6H5NO2 Negative 20 Nicotinate 12.41 122.02 78.03318

124.0069 C2H7NO3S Negative 20 Taurine 10.03 124.01 79.95557

128.0348 C5H7NO3 Negative 23 Pyroglutamic acid 10.09 128.04 82.02811

129.0188 C5H6O4 Negative 25 Citraconic acid 9.61 129.02 85.02773

129.0552 C6H10O3 Negative 20 2-Ketohexanoic acid 2.45 129.06 84.94134

130.0504 C5H9NO3 Negative 20 N-Acetyl-L-alanine 11.95 130.05 88.03863

130.9981 C4H4O5 Negative 20 Oxalacetic acid 10.87 131 87.04337

131.0702 C6H12O3 Negative 20 Hydroxyisocaproic acid 10.21 131.07 85.02769

133.0137 C4H6O5 Negative 20 Malic acid 11.35 133.01 115.002

135.0307 C5H4N4O Negative 20 Hypoxanthine 7.59 135.03 92.02372

136.0399 C7H7NO2 Negative 20 p-Aminobenzoic acid 7.6 136.04 92.02366

139.9749 CH4NO5P Negative 24 Carbamoylphosphate 13.27 139.98 78.95741

145.0137 C5H6O5 Negative 20 Oxoglutaric acid 10.11 145.01 101.0227

145.029 C9H6O2 Negative 22 Phenylpropiolic acid 10.11 145.03 101.0227

146.1322 C5H10O3N Negative 40 13C-Glutamine 12.18 146.13 128.0531

147.0116 C5H8O3S Negative 20 2-oxo-4-methylthiobutanoate 12.08 147.01 103.0577

147.0294 C5H8O5 Negative 20 Ribonolactone 8.49 147.01 59.01213

147.0294 C5H8O5 Negative 20 (S)-2-Hydroxyglutarate 12.18 147.03 103.0577

149.0603 C9H10O2 Negative 21 3-methylphenylacetic acid 2.88 149.06 105.0693

149.0603 C9H10O2 Negative 21 2-Phenylpropionate 2.78 149.06 105.0693

151.0256 C5H4N4O2 Negative 23 Xanthine 8.02 151.03 108.0187

151.0395 C8H8O3 Negative 26 Hydroxyphenylacetic acid 2.35 151.04 107.0487

153.0188 C7H6O4 Negative 20 2,3-Dihydroxybenzoate 1.73 153.02 109.0279

155.0093 C5H4N2O4 Negative 20 Orotic acid 9.1 155.01 111.0183

157.025 C5H6N2O4 Negative 20 L-Dihydroorotic acid 9.67 157.03 113.034

157.0362 C4H6N4O3 Negative 20 Allantoin 8.05 157.04 97.00268

160.0399 C9H7NO2 Negative 32 Indole-3-carboxylic acid 9.48 160.04 74.02298

160.061 C6H11NO4 Negative 20 L-2-Aminoadipic acid 12.27 160.06 142.0494

163.0395 C9H8O3 Negative 20 Phenylpyruvate 2.25 163.04 91.05362

165.0552 C9H10O3 Negative 22 Phenyllactic acid 2.87 165.06 119.0485

165.0552 C9H10O3 Negative 24 Quinolinic acid 9.73 165.06 72.99592

166.0141 C7H5NO4 Negative 20 Phosphoenolpyruvic acid 11.46 166.01 122.0877

166.9746 C3H5O6P Negative 22 Uric acid 12.21 166.98 78.95729

167.0205 C5H4N4O3 Negative 21 D-Glyceraldehyde 3-phosphate 10.67 167.02 124.0137

168.9902 C3H7O6P Negative 20 Glycerol 3-phosphate 12.15 168.99 96.96792

168.9902 C3H7O6P Negative 40 Dihydroxyacetone phosphate 11.91 168.99 96.96794

173.0086 C6H6O6 Negative 20 Trans-Aconitic acid 10.12 173.01 85.02775

173.045 C7H10O5 Negative 20 Shikimate 1.7 173.05 93.03284

175.0243 C6H8O6 Negative 34 Ascorbic acid 10.8 175.02 71.01209

175.0355 C5H8N2O5 Negative 20 Ureidosuccinic acid 11.86 175.04 132.0287

175.0468 C4H8N4O4 Negative 20 Allantoate 11.11 175.05 89.04192

175.0607 C7H12O5 Negative 20 2-Isopropylmalic acid 11.11 175.06 89.04189

176.9354 H4O7P2 Negative 31 Pyrophosphate 12.48 176.94 78.95727

177.0399 C6H10O6 Negative 20 Gluconolactone 11.86 177.04 134.0332

179.0345 C9H8O4 Negative 20 4-hydroxyphenylpyruvate 13.33 179.03 87.00706

179.0556 C6H12O6 Negative 20 Myo-inositol 13.16 179.06 87.00703

179.0708 C7H6O3 Negative 23 p-Salicylic acid 2.31 179.07 93.0329

182.0123 C4H9NO5S Negative 23 L-homocysteic acid 2.67 182.01 79.95565

182.0454 C8H9NO4 Negative 23 4-Pyridoxic acid 1.72 182.05 138.0181

184.9851 C3H7O7P Negative 20 3-Phosphoglyceric acid 9.77 184.99 78.95724

186.0167 C3H8NO6P Negative 20 Phosphoserine 9.77 184.0011 78.95725

186.0555 C11H9NO2 Negative 20 Indoleacrylic acid 8.41 186.06 142.065

188.0348 C10H7NO3 Negative 21 Kynurenic acid 11.26 188.03 144.0652

191.0192 C6H8O7 Negative 20 Citrate 12.49 186.02 111.0071

191.0192 C6H8O7 Negative 20 Isocitrate 12.49 186.02 111.0071

193.0349 C6H10O7 Negative 20 D-Glucuronic acid 11.88 193.04 113.0228

195.0505 C6H12O7 Negative 20 Gluconic acid 11.36 195.05 75.00703

199.0008 C4H9O7P Negative 20 D-Erythrose 4-phosphate 10.52 199 96.96774

204.0297 C10H7NO4 Negative 20 Xanthurenic acid 9.72 204.03 116.0489

209.0298 C6H10O8 Negative 20 d-Glucarate 12.26 209.03 165.0181

213.0164 C5H11O7P Negative 35 Deoxyribose 5-phosphate 2.95 213.02 96.95845

218.1029 C9H17NO5 Negative 21 Pantothenate 9.29 218.1 88.03867

225.0399 C10H10O6 Negative 20 Prephenate 13.9 225.04 110.0708

227.0668 C9H12N2O5 Negative 20 Deoxyuridine 1.76 227.07 112.984

229.0114 C5H11O8P Negative 20 Xylulose 5-phosphate 12.22 229.01 96.96778

243.027 C6H13O8P Negative 32 Fucose 1-phosphate 12.54 243.03 96.96791

243.0617 C9H12N2O6 Negative 21 Uridine 7.24 243.06 110.0231

251.0781 C10H12N4O4 Negative 21 Deoxyinosine 10.08 251.08 124.0058

257.0063 C6H11O9P Negative 20 6-Phosphonoglucono-D-lactone 2.77 257.01 195.1748

259.0219 C6H13O9P Negative 20 D-Fructose 6-phosphate 12.48 259.02 96.96791

259.0219 C6H13O9P Negative 20 D-Glucose 6-phosphate 12.48 259.02 96.96791

259.0219 C6H13O9P Negative 20 1D-myo-Inositol 3-phosphate 12.54 259.02 241.0118

259.0219 C6H13O9P Negative 20 D-Myoinositol 4-phosphate 11.3 259.02 213.0512

259.0219 C6H13O9P Negative 20 D-Mannose 6-phosphate 12.92 259.02 199.0006

259.0219 C6H13O9P Negative 20 D-Fructose 1-phosphate 12.48 259.02 168.9896

261.0375 C6H13O9P Negative 20 Glucose-1-phosphate 16.79 259.02 241.0116

264.9515 C3H8O10P2 Negative 20 2,3-Diphosphoglyceric acid 12.4 264.95 78.95732

267.073 C10H12N4O5 Negative 27 Inosine 8.93 267.07 135.0297

275.0168 C6H13O10P Negative 20 6-Phosphogluconic acid 12.86 275.02 96.9679

283.0679 C10H12N4O6 Negative 24 Xanthosine 10 283.07 151.0248

287.0516 C10H12N2O8 Negative 20 Orotidine 10.06 287.0516 111.0184

289.0325 C7H15O10P Negative 27 Sedoheptulose 7-phosphate 12.68 289.03 96.9679

300.0485 C8H16NO9P Negative 20 N-Acetyl-alpha-D-glucosamine 1-phosphate 12.31 300.05 96.96791

306.076 C10H17N3O6S Negative 20 Glutathione 12.06 306.08 143.0448

307.0332 C9H13N2O8P Negative 20 dUMP 12.06 307.03 143.0448

313.0606 C10H20O7P2 Negative 20 Geranyl diphosphate 12.13 313.06 78.95132

321.0488 C10H15N2O8P Negative 22 dTMP 11.2 321.05 195.0057

328.0447 C10H12N5O6P Negative 33 cAMP 11.2 328.04 134.0458

338.9882 C6H14O12P2 Negative 30 Fructose 1,6-bisphosphate 13.25 338.99 96.96793

341.1084 C12H22O11 Negative 20 Trehalose 13 341.11 142.9737

367.0179 C10H13N2O11P Negative 20 Orotidylic acid 1.68 367.02 96.9582

381.1232 C15H28O7P2 Negative 22 Farnesyl pyrophosphate 1.6 381.1232 96.95839

383.1138 C14H20N6O5S Negative 31 S-adenosyl-L-homocysteine 12.76 383.11 134.0458

386.0155 C9H15N3O10P2 Negative 27 dCDP 2.11 386.02 96.95841

388.944 C5H13O14P3 Negative 20 Phosphoribosyl pyrophosphate 12.33 389.94 96.95844

401.0151 C10H16N2O11P2 Negative 27 CDP 12.36 402.02 96.95847

402.0104 C9H15N3O11P2 Negative 22 dTDP 12.33 402.01 96.9584

402.9944 C9H14N2O12P2 Negative 28 Uridine 5'-diphosphate 12.89 402.99 158.924

407.2798 C24H40O5 Negative 42 Cholic acid 2.21 407.28 57.07066

421.0747 C12H23O14P Negative 36 Sucrose-6-phosphate 12.55 421.07 78.95734

421.0747 C12H23O14P Negative 36 Lactose 6-phosphate 12.52 421.07 78.95729

421.0747 C12H23O14P Negative 36 Trehalose-6-Phosphate 12.52 421.07 78.95729

423.0293 C12H18N4O7P2S Negative 21 Thiamine pyrophosphate 1.56 423.03 341.1028

426.0216 C10H15N5O10P2 Negative 27 ADP 13.19 426.02 158.9241

427.0056 C10H14N4O11P2 Negative 27 IDP 13.24 427.01 158.9237

442.0165 C10H15N5O11P2 Negative 21 Guanosine diphosphate 12.62 442.02 152.0564

455.0968 C17H21N4O9P Negative 21 FMN 11.45 455.1 96.96799

465.3039 C27H46O4S Negative 22 Cholesteryl sulfate 1.62 465.3 96.95844

465.9818 C9H16N3O13P3 Negative 30 dCTP 1.43 465.98 96.95836

466.9658 C9H15N2O14P3 Negative 27 Deoxyuridine triphosphate 12.48 466.97 78.95731

480.9815 C10H17N2O14P3 Negative 33 Thymidine 5'-triphosphate 12.94 480.98 158.9242

481.9767 C9H16N3O14P3 Negative 24 Cytidine triphosphate 2.56 481.98 307.2643

482.9607 C9H15N2O15P3 Negative 35 Uridine triphosphate 13.41 482.96 158.9241

505.9879 C10H16N5O13P3 Negative 30 Adenosine triphosphate 13.26 505.99 158.9241

565.0472 C15H24N2O17P2 Negative 25 UDP-glucose 12.44 565.05 323.0287

579.0265 C15H22N2O18P2 Negative 26 Uridine diphosphate glucuronic acid 12.91 579.03 402.9946

588.0744 C16H25N5O15P2 Negative 24 ADP-glucose 12.9 588.07 442.0164

Table S2. VIP scores and quantification result of the top 15 metabolites in the T2DM PLS-DA model. Significance was determined

by the Student’s t-test.

Metabolite names Comp. 1 Comp. 2 Comp. 3 Comp. 4 Comp. 5 Ratio(T2DM/CTL) p-Value

Shikimate 13.741 8.8962 8.2985 7.7637 7.7022 3.17 0.0055

Deoxyuridine 2.4885 5.9968 6.4602 6.2601 6.2111 4.76 0.0000

DL-Pipecolic acid 2.6145 5.586 5.3324 5.372 5.3301 0.39 0.0003

Urea 1.8152 5.2995 5.1298 4.928 4.8913 1.46 0.0014

Ornithine 1.1385 3.7663 3.7876 3.5487 3.6792 1.30 0.0958

Valine 1.077 2.8499 2.9899 3.7943 3.7674 1.12 0.1090

5methoxytryptophan 1.0851 2.2381 2.3614 2.8634 2.9526 0.47 0.0150

2-keto-isovalerate 0.59014 1.474 1.6758 1.6529 1.6498 2.78 0.0000

Proline 0.31391 1.3989 2.543 3.5161 3.5485 1.05 0.6689

N-acetylornithine 0.46273 1.269 1.4407 1.355 1.3479 1.16 0.0010

Creatinine 0.52225 0.88265 0.824 0.77532 0.88213 1.26 0.0152

Guanosine triphosphate 0.30208 0.69213 0.79947 0.77698 0.80172 1.71 0.0005

2-ketohaxanoic acid 0.060388 0.5703 0.99262 1.0189 1.0541 0.94 0.7132

Taurine 0.17343 0.40162 0.38645 0.47725 0.52139 0.77 0.0908

L-carnitine 0.1743 0.38828 0.45656 0.44667 0.44317 0.62 0.0040