Supplementary Data Metabolomics Combined with Pattern Recognition

Electronic Supplementary Material (ESI) for Molecular BioSystems. This journal is © The Royal Society of Chemistry 2017

Supplementary data

Metabolomics Combined with Pattern Recognition and Bioinformatics Analysis

Methods for the Pharmacodynamic Biomarkers Development on Liver Fibrosis

Junwei Fang1#, Liping Wang2#, Yang Wang1, Mingfeng Qiu3*, Yongyu Zhang1,4*

1Center for Traditional Chinese Medicine and Systems Biology, Shanghai University of

Traditional Chinese Medicine, 1200 Cailun Road,Shanghai 201203,P.R. China

2School of Pharmacy, Fudan University, 826 Zhangheng Road,Shanghai 201203,P.R.China

3School of Pharmacy, Shanhai Jiao Tong University, Shanghai 200240, P.R. China

4School of Traditional Dai Medicine, West Yunnan University of Applied Sciences, Jinghong

600100, P.R. China

*To whom correspondence should be address.

E-mail: [email protected]

Tel.: +86 21 13817553148

Fax: +86 21 51322642. Fig. S1. Retention time deviation profile generated by XCMS. The retention time deviation was < ± 25 s for GC-EI-MS analyses. 100 3 SD

2 SD 50 QC-1 QC-2 QC-3 QC-4 QC-5 ] 1 [

t 0 QC-6 QC-7 QC-8 QC-9 -50 QC-10 2 SD -100 3 SD 1 2 3 4 5 6 7 8 9 10 Num

(A)

60 3 SD

40 2 SD QC-6 QC-7 20 QC-8 ]

2 QC-2 QC-9 [

t 0 QC-1 QC-3 QC-4 -20 QC-5 QC-10 -40 2 SD -60 3 SD 1 2 3 4 5 6 7 8 9 10 Num

(B) Fig. S2. PCA first and second component for QC samples versus time analyzed. X-axis: run order; Y-axis: standard deviation. All the 10 inserted QC samples are distributed within 2 SD regions. (A) Component 1 versus numbers of the QC samples; (B) Component 2 versus numbers of the QC samples. Table S1 Metabolites identified by searching NIST and Fiehn databases (with minimum match factor > 70%) Rentime Weighted No. HMDB Name (min) Probability 1 2-Methylpropanoic acid 5.3384 88 2 Methylamine 5.3514 86 3 Hydrogen sulfide 6.3016 82 4 N-Methylhydantoin 6.3217 71 5 1,2,4-Trimethylbenzene 6.6884 90 6 Ethylene glycol 6.7602 94 7 Ammonia 7.6413 74 8 6-Hydroxynicotinic acid 7.9977 97 9 Phenol 8.5313 92 10 L-lactic acid 8.629 97 11 2-hydroxybutyric acid 8.7396 87 12 Hexanoic acid 9.0954 71 13 glycolic acid 9.1232 97 14 Pyruvic acid 9.4783 94 15 L-alanine 9.9842 95 16 Hydroxylamine 10.3679 93 17 fumaric acid 10.7721 81 18 oxalic acid 11.0315 96 19 2-furoic acid 11.1874 76 20 Propylene glycol 11.4662 75 21 p-cresol 11.7618 98 22 2-Oxobutanoate 12.0723 88 23 3-Aminoisobutyric acid 13.3459 72 24 L-mimosine 13.5309 88 25 Methylmalonic acid 14.2852 73 26 2-Ethylhydracrylic acid 14.8998 88 27 benzoic acid 15.6611 96 28 (R)-3-Hydroxybutanoate 16.2297 87 29 2-Hydroxyphenethylamine 16.4742 83 30 Octanoic acid 16.4962 95 31 phosphoric acid 17.1094 88 32 nicotinic acid 17.1469 81 33 Malonate 17.2549 95 34 L-Isoleucine 17.5966 83 35 glycerol 17.7021 76 36 L-proline 17.85 92 37 Benzeneacetic acid 17.9581 87 38 glycine 18.2628 97 39 4-Methyl-2-oxopentanoate 18.4186 87 40 2,5-Dihydroxypyridine 18.592 71 41 catechol 18.6399 90 42 succinic acid 18.7984 94 43 5-Hydroxyhexanoic acid 19.3171 71 44 glyceric acid 19.5416 92 45 uracil 19.7378 96 46 Dihydrouracil 19.7408 87 47 Citraconic acid 20.3347 97 48 4-Deoxyerythronic acid 20.5345 71 49 6-Hydroxyhexanoic acid 20.7474 90 50 pipecolic acid 20.8399 97 51 L-serine 20.961 95 52 thymine 21.0999 79 53 L-threonine 22.137 96 54 hydroquinone 22.7779 86 55 Pentanedioic acid 23.1828 85 56 2,4-Dihydroxybutanoic acid 23.4946 85 57 Ethanolamine 23.9927 73 58 3,4-Dihydroxybutanoic acid 24.5231 94 59 Glutaconic acid 24.7282 74 60 (R)-Citramalic acid 26.095 88 61 p-Synephrine 26.1991 85 62 D-malic acid 27.1274 91 63 D-Threitol 27.7658 73 64 adipic acid 27.8881 95 65 D-threitol 28.1041 95 66 L-glutamic acid 28.2395 92 67 L-pyroglutamic acid 28.3047 93 68 cytosine 28.6533 89 69 4-Hydroxy-L-proline 28.6986 76 70 4-guanidinobutyric acid 28.7581 89 71 1,2,3-Trihydroxybenzene 29.0473 70 72 Creatinine 29.6598 91 73 2,3,4-Trihydroxybutanoic acid 29.6747 86 74 L-Cysteine 29.9781 77 75 Mevalonic acid 30.2731 83 76 4-hydroxybenzoic acid 30.4639 84 77 alpha ketoglutaric acid 31.1716 76 78 Heptanedioic acid 31.3488 82 79 Phenylethylamine 31.7576 72 80 hypotaurine 31.7609 93 81 benzene-1,2,4-triol 32.0261 97 82 3-Hydroxymethylglutaric acid 32.2441 96 83 L-Glutamine 33.0994 79 84 4-hydroxyphenylacetic acid 33.748 85 85 D-lyxose 34.2869 96 86 Iso-Valeraldehyde 34.9305 78 87 fructose 35.5219 74 88 3-Hydroxyadipic acid 35.7746 87 89 Octanedioic acid 36.2837 73 90 xylitol 36.6214 97 91 Ribitol 37.1182 78 92 D-Galactose 37.1382 86 93 putrescine 37.3206 95 94 L-fucose 37.5186 83 95 L-arabinopyranose 37.5634 73 96 Valproic acid 37.5859 75 97 orotic acid 37.8253 93 98 cis-aconitic acid 38.1072 93 99 L-Tyrosine 38.1773 78 100 4-hydroxy-3-methoxybenzoic acid 38.3241 85 101 2-Keto-D-gluconic acid 38.4591 91 102 glycerol 1-phosphate 38.5846 86 103 Ribonolactone 38.747 83 104 4-Hydroxymandelate 38.9191 92 105 Azelaic acid 39.4527 77 106 5-hydroxy-L-tryptophan 39.6597 77 107 citric acid 40.1015 94 108 isocitric acid 40.2231 94 109 Cadaverine 40.3072 85 110 3,4-Dihydroxymandelate 40.4212 85 111 homogentisic acid 40.4579 75 112 Indole-2-carboxylic acid 40.5157 75 113 Tartaric acid 40.621 72 114 Dehydroascorbic acid 40.6455 83 115 Myristic acid 40.8564 91 116 hippuric acid 40.9924 97 117 tagatose 41.0679 84 118 adrenaline 41.5688 93 119 gluconic acid lactone 41.5997 88 120 Indole 41.6462 73 121 D-glucose 41.7976 95 122 Myo-Inositol 41.8818 72 123 D-allose 42.3869 92 124 D-mannose 42.4808 95 125 L-Gulonolactone 42.6799 93 126 Mannitol 42.8158 96 127 galacturonic acid 42.918 89 128 Sedoheptulose 42.973 74 129 D-sorbitol 43.0816 91 130 Ascorbic acid 43.2291 75 131 Indole-3-acetate 43.44 81 132 gluconic acid 43.5758 91 133 5-Aminolevulinic acid 43.8236 73 134 Pantothenic acid 44.2091 81 135 Galactonic acid 44.3061 90 136 D-saccharic acid 44.5823 96 137 D-Glucuronic acid 44.9808 91 138 mucic acid 45.3439 98 139 palmitic acid 45.4479 96 140 sebacic acid 45.8127 78 141 N-Acetyl-D-mannosamine 46.0572 90 142 ferulic acid 46.3504 90 143 Uric acid 46.488 97 144 Glucosamine 46.7079 78 145 Elaidic acid 48.7132 90 146 5-Hydroxyindoleacetaldehyde 48.7753 93 147 Oleic acid 48.8529 92 148 stearic acid 49.375 94 149 xanthurenic acid 49.5202 71 150 Pseudouridine 51.0723 75 151 Galactitol 51.9406 78 152 myo-Inositol 1-phosphate 52.4289 74 153 arachidic acid 53.2561 94 154 Sucrose 59.1188 93 155 behenic acid 59.3852 96 156 Alpha-Lactose 61.9974 89 Table S2 Difference metabolites related to CCl4-induced liver fibrosis in rats Fold change No. Metabolite Rentime(min) VIP P value (Model/Normal) 1 2-Methylpropanoic acid 5.34 1.05 0.037 0.52 2 N-Methylhydantoin 6.32 1.51 0.001 0.45 3 1,2,4-Trimethylbenzene 6.69 1.44 0.001 0.35 4 L-lactic acid 8.63 1.42 0.001 0.44 5 Hexanoic acid 9.10 1.57 0.000 0.36 6 glycolic acid 9.12 1.33 0.004 0.60 7 Pyruvic acid 9.48 1.09 0.027 0.59 8 L-alanine 9.98 1.05 0.036 0.71 9 oxalic acid 11.03 1.58 0.000 2.46 10 2-furoic acid 11.19 1.65 0.000 0.22 11 Propylene glycol 11.47 1.13 0.020 0.66 12 3-Aminoisobutyric acid 13.35 1.51 0.000 8.03 13 2-Ethylhydracrylic acid 14.90 1.28 0.007 0.54 14 benzoic acid 15.66 1.03 0.041 0.51 15 (R)-3-Hydroxybutanoate 16.23 1.16 0.023 0.64 16 Octanoic acid 16.50 1.57 0.000 0.21 17 phosphoric acid 17.11 1.07 0.029 1.85 18 Benzeneacetic acid 17.96 1.08 0.028 0.53 19 succinic acid 18.80 1.08 0.028 1.98 20 glyceric acid 19.54 1.61 0.000 0.11 21 4-Deoxyerythronic acid 20.53 1.38 0.003 5.93 22 L-threonine 22.14 1.51 0.000 4.75 23 Pentanedioic acid 22.18 1.20 0.012 2.16 24 (R)-Citramalic acid 26.10 1.38 0.002 2.22 25 p-Synephrine 26.20 1.34 0.003 0.12 26 D-malic acid 27.13 1.44 0.001 5.82 27 L-glutamic acid 28.24 1.16 0.016 2.40 28 L-pyroglutamic acid 28.30 1.34 0.004 2.67 29 cytosine 28.65 1.32 0.004 5.64 30 L-Cysteine 29.98 1.07 0.031 2.09 31 alpha ketoglutaric acid 31.17 1.57 0.000 14.92 32 Phenylethylamine 31.76 1.52 0.001 5.48 33 hypotaurine 31.76 1.52 0.001 5.49 34 3-Hydroxymethylglutaric acid 32.24 1.62 0.000 3.30 35 3-Hydroxyadipic acid 35.77 1.60 0.000 3.29 36 xylitol 36.62 1.41 0.003 1.87 37 L-arabinopyranose 37.56 1.44 0.001 0.46 38 Valproic acid 37.59 1.42 0.001 0.46 39 orotic acid 37.83 1.57 0.000 6.68 40 cis-aconitic acid 38.11 1.43 0.002 6.06 41 4-hydroxy-3-methoxybenzoic acid 38.32 1.36 0.003 3.42 42 2-Keto-D-gluconic acid 38.46 1.41 0.002 0.57 43 Ribonolactone 38.75 1.31 0.005 0.45 44 5-hydroxy-L-tryptophan 39.66 1.33 0.004 0.26 45 citric acid 40.10 1.39 0.002 3.49 46 isocitric acid 40.22 1.30 0.005 26.89 47 homogentisic acid 40.46 1.29 0.006 0.42 48 Dehydroascorbic acid 40.65 1.68 0.000 0.38 49 adrenaline 41.57 1.16 0.016 0.65 50 gluconic acid lactone 41.60 1.25 0.008 0.61 51 Ascorbic acid 43.23 1.65 0.000 0.08 52 gluconic acid 43.58 1.74 0.000 0.20 53 palmitic acid 45.45 1.47 0.001 0.04 54 sebacic acid 45.81 1.07 0.031 3.16 55 ferulic acid 46.35 1.41 0.002 9.10 56 Elaidic acid 48.71 1.35 0.003 0.03 57 5-Hydroxyindoleacetaldehyde 48.78 1.32 0.005 2.60 58 Oleic acid 48.85 1.20 0.013 0.03 59 stearic acid 49.38 1.71 0.000 0.11 60 xanthurenic acid 49.52 1.09 0.035 12.32 61 Galactitol 51.94 1.38 0.004 2.74 62 myo-Inositol 1-phosphate 52.43 1.06 0.047 6.69 63 Sucrose 59.12 1.07 0.036 9.84 Note: VIP means the variable importance in the project; P value was obtained with Student’s t test. Table S3 MetaboAnalyst 3.0 identified highly significant enriched pathways (p<0.05). No. Metabolic pathway Total Hits Raw P value -log(p) FDR 1 Glyoxylate and dicarboxylate metabolism 16 5 3.140E-04 8.07 2.540E-02 2 Citrate cycle (TCA cycle) 20 5 9.800E-04 6.93 3.970E-02 3 Butanoate metabolism 20 4 7.780E-03 4.86 2.100E-01 4 Alanine, aspirate and glutamate metabolism 24 4 1.510E-02 4.19 3.050E-01 5 Glycine, scrine and threonine metabolism 32 4 3.990E-02 3.22 5.420E-01 6 Taurine and hypotaurine metabolism 8 2 4.010E-02 3.22 5.420E-01 Note: The Total is the total number of compounds in the pathway; the Hits are the actually matched number of metabolites from the user uploaded data; the Raw p value: the original p value calculated from the enrichment analysis; the FDR p is the p value adjusted using False Discovery Rate.