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Supplementary materials

Serum pretreatment for CE-MS analysis

50 μL of aliquot serum was mixed with 450 μL of methanol containing 10 μM internal standards (L- sulfone and D-camphor-10-sulfonic acid sodium salt), and 500 μL of chloroform was added into the mixture and vortexed again. Then,

200 μL of water was added to form a two-phase system. After 5 min standing, the mixture was centrifuged at 5,000 g for 10 min at 4 °C. Subsequently, 420 μL of the supernatant was transferred and centrifugally filtered through a 5 kDa cutoff filter to remove proteins. The filtrate was lyophilized and dissolved with water.

Detailed conditions of capillary electrophoresis and mass spectrometry for betaine and propionylcarnitine analysis

CE-MS experiment was performed on a CE (G7100A, Agilent, USA)-TOF/MS

(G6224A, Agilent). An 80 cm length and 50 μm inner diameter fused silica capillary was used for the separation. Capillary temperature was maintained at 20 °C, and sample manager temperature was set at 5 °C. CE-MS coupling was realized by a coaxial sheath liquid interface with methanol/water (50% v/v) and 0.1 μM hexakis (2,

2-difluoroethoxy) phosphazene at 10 μL/min flow.

Background electrolyte was 1 M formic acid in cation-positive mode.

Approximately 3 nL sample solution was injected at 50 mbar for 3 s, and a voltage of

27 kV was applied. The electrospray of ESI-TOF/MS was performed in the positive mode at 4 kV. Other parameters were set as follows: Dry gas temperature, 300 °C;

Nitrogen flow, 7 L/min; Nebulizer pressure, 5 psig; Fragmentor, skimmer, and Oct

RFV voltages were 105, 50, and 650 V, respectively. The protonated isotope of 2 / 13 methanol dimer (i.e., [2MeOH + H]+, m/z 66.0631) and protonated hexakis (2,

2-difluoroethoxy) phosphazene (m/z 622.0289) were used as reference masses to provide the lock mass for real-time calibration of exact mass measurement. The exact mass data were acquired at a rate of 1.5 spectra/s from 100 to 1000 m/z.

Table S1 Clinical characteristics of the patients with liver diseases for biomarker discovery from serum samples Samples Hepatitis Cirrhosis HCC Sample No. 81 78 139 Age (yr) 44 (24~58) 47 (22~71) 51.7 (30~78) Sex (F/M) 26/55 24/54 22/117 AFP>20 (μg/L) 31 25 87 ALT (U/L) 175.6 (18.2~1588.5) 74.6 (4.3~883.0) 69.6 (11.1~700.9) AST (U/L) 104.9 (19.3~389.1) 81.3 (10.1~557.3) 67.9 (13.1~669.4) HBsAg-positive/negative 81/0 78/0 117/22 anti-HCV-positive/negative 0/81 0/78 0/139 Tumor diameter (cm) 5.31 (0.9~20)

Table S2 Clinical characteristics of the cirrhosis patients and HCC patients for external validation Samples Cirrhosis HCC Sample No. 25 22 Age (yr) 53.4 (34-72) 53.5 (21-66) Sex (Female/Male) 18/7 18/4 AFP>20(μg/L) 11 ALT (U/L) 67.7 (11.7~297) 102.2 (29~636) AST (U/L) 77.3 (13.8~500) 81.3 (26~226) Tumor diameter (cm) 7.7 (3.2~14.8) 1 Ⅱ

12 Ⅲ Edmonson stage 1 Ⅱ+Ⅲ

Not determined 8 3 / 13

Table S3 Identification results of significant metabolites between carcinoma tissue and distal noncancerous tissue

a tR(min) m/z Metabolite p Value Change Pathway involved ESI+ 0.696 258.110 Glycerophosphocholine* <0.001 0.404 Glycerophospholipid metabolism Lysophospholipase 0.727 156.042 Betaine* <0.001 0.562 Glycine, serine and threonine Choline oxidase 0.764 162.113 Carnitine* 0.001 0.753 Fatty acid metabolism Palmitoyl-CoA 0.806 175.002 Hypoxanthine* 0.027 1.204 Purine metabolism Xanthine Phosphoethanolamine 0.827 184.074 Phosphorylcholine <0.001 2.556 Glycerophospholipid metabolism N-methyltransferase 0.864 204.123 Acetylcarnitine* <0.001 1.869 Fatty acid metabolism Acetyl-CoA 0.906 145.050 3-Hexenedioic acid <0.001 0.540 Fatty acid metabolism 0.978 150.059 Methionine* 0.017 1.200 and methionine metabolism Coproporphyrinogen dehydrogenase 0.979 262.129 Methylglutarylcarnitine 0.003 0.563 Biosynthesis of unsaturated fatty acids Methyl glutaryl-CoA 0.986 269.089 Arabinosylhypoxanthine 0.008 0.804 Purine metabolism Oxygen 5'- 0.989 123.055 Niacinamide* <0.001 0.632 Nicotinate and nicotinamide metabolism Nicotinamide N-methyltransferase 0.989 248.150 Hydroxybutyrylcarnitine 0.011 0.709 1.031 276.145 Glutarylcarnitine 0.016 0.507 Biosynthesis of unsaturated fatty acids 1.037 218.139 Propionylcarnitine* <0.001 0.519 Beta-oxidation of pristanoyl-CoA Pristanoyl-CoA 1.14 250.094 S-Acetyldihydrolipoamide <0.001 0.505 Alanine, aspartate and pyruvate metabolism Pyruvate dehydrogenase 1.202 384.115 Succinyladenosine <0.001 0.327 Dephosphorylated enzyme 1.326 688.157 Dephospho-CoA <0.001 0.213 Pantothenate and CoA biosynthesis Dephospho-CoA kinase 1.77 232.155 Isobutyryl-L-carnitine 0.004 1.682 Fatty acid metabolism Acyl-CoA 1.96 298.097 5'-Methylthioadenosine* <0.001 4.700 Cysteine and methionine metabolism MTA phosphorylase 2.212 276.181 Hydroxyhexanoycarnitine 0.003 0.542 4 / 13

2.987 229.155 Isoleucyl-L- 0.009 0.697 6.088 260.186 Hexanoylcarnitine <0.001 0.462 Fatty acid metabolism Carnitine Palmitoyl transferase 8.736 286.202 2-Octenoylcarnitine <0.001 0.384 10.823 591.319 Urobilinogen 0.001 0.148 Porphyrin and chlorophyll metabolism Oxygen oxidoreductase 13.279 450.322 Glycoursodeoxycholic acid* 0.004 0.409 Bile acid biosynthesis Bile acid-CoA 13.385 466.317 Glycocholic acid* 0.002 0.565 Bile acid biosynthesis Bile acid-CoA 14.531 450.322 Glycodeoxycholic acid* 0.030 0.580 Bile acid biosynthesis Choloylglycine hydrolase 15.312 300.29 Palmitoylethanolamide* 0.003 0.287 Lipid metabolism 15.816 520.341 LPC C18:2 <0.001 0.250 Glycerophospholipid metabolism Lysophospholipid acyltransferase 16.18 542.326 LPC C20:5 0.018 0.416 Glycerophospholipid metabolism Lysophospholipid acyltransferase 16.233 478.294 LPE C18:2 0.001 0.378 Lysophospholipid metabolism Phospholipase A 16.277 502.294 LPE C20:4 0.001 0.387 Lysophospholipid metabolism Phospholipase A 16.277 526.294 LPE C22:6 <0.001 0.242 Lysophospholipid metabolism Phospholipase A 16.277 544.341 LPC C20:4 <0.001 0.310 Glycerophospholipid metabolism Lysophospholipid acyltransferase 16.278 568.341 LPC C22:6 <0.001 0.197 Glycerophospholipid metabolism Lysophospholipid acyltransferase 16.299 454.293 LPE C16:0 0.011 0.534 Lysophospholipid metabolism Phospholipase A 16.394 520.341 LPC C18:2* <0.001 0.280 Glycerophospholipid metabolism Lysophospholipid acyltransferase 16.71 496.340 LPC C16:0* 0.003 0.500 Glycerophospholipid metabolism Lysophospholipid acyltransferase 17.031 522.356 LPC C18:1 0.033 0.546 Glycerophospholipid metabolism Lysophospholipid acyltransferase 17.676 524.372 LPC C18:0* 0.049 0.620 Glycerophospholipid metabolism Lysophospholipid acyltransferase 21.332 756.555 PC C34:3 0.001 0.412 21.389 780.554 PC C36:5 0.002 0.409 21.601 806.570 PC C38:6 <0.001 0.305

ESI- 0.623 611.142 disulfide* 0.004 5.037 Glutathione metabolism Glutathione reductase 5 / 13

0.627 275.016 6-Phosphogluconic acid <0.001 4.369 Pentose phosphate pathway Phosphogluconate 2-dehydrogenase; 0.635 171.006 Glycerol 3-phosphate <0.001 0.360 Glycerophospholipid metabolism Glycerol-3-phosphate dehydrogenase 0.645 333.058 Gycerophosphoinositol <0.001 2.183 0.647 338.987 Fructose 1,6-bisphosphate* <0.001 5.212 Glycolysis metabolism Pyruvate kinase 0.648 133.014 Malate* <0.001 0.563 Tricarboxylic acid cycle Malic dehydrogenase 0.648 147.029 Citramalic acid 0.048 2.472 Tricarboxylic acid cycle Citramalate CoA-transferase 0.649 308.097 N-Acetylneuraminic acid <0.001 0.466 0.65 115.003 Fumarate* 0.008 0.766 Tricarboxylic acid cycle Fumarate reductase 0.654 175.024 Ascorbic acid 0.001 1.975 Glutathione metabolism L-gulonolactone oxidase 0.655 259.021 Glucose-6-phosphate* 0.004 2.242 Glycolysis metabolism Glycosyltransferases Phosphoethanolamine 0.656 140.012 O-Phosphoethanolamine* <0.001 2.198 Glycerophospholipid metabolism N-methyltransferase 0.671 225.061 Glucoheptonic acid 0.003 0.721 0.672 130.051 N-Acetyl-L-* 0.008 1.572 and proline metabolism Amino-acid N-acetyltransferase 0.683 242.079 Cytidine <0.001 0.549 Pyrimidine metabolism Pyrimidine-nucleoside phosphorylase 0.686 102.056 Gamma-Aminobutyric acid* 0.037 1.224 Alanine, aspartate and glutamate metabolism Aldehyde dehydrogenase 0.691 132.030 Aspartate* 0.011 1.433 Alanine, aspartate and glutamate metabolism Aspartate dehydrogenase Glycerylphosphorylethanolami 0.693 214.048 0.023 0.719 Glycerophospholipid metabolism Lysophospholipase ne 0.714 124.007 Taurine* <0.001 1.768 Bile acid biosynthesis Bile acid-CoA 0.769 306.075 Glutathione 0.029 2.337 Glutathione metabolism Glutathione reductase 0.778 104.035 * 0.002 1.486 , serine and metabolism Serine 3-dehydrogenase 0.778 118.051 Threonine 0.007 1.308 Glycine, serine and threonine metabolism Threonine 3-dehydrogenase Succinate-semialdehyde 0.788 117.019 Succinate* <0.001 0.305 Tricarboxylic acid cycle dehydrogenase 0.805 167.020 Uric acid* 0.006 0.783 Purine metabolism Uric acid oxidase 6 / 13

0.826 151.026 Xanthine* <0.001 0.659 Purine metabolism Xanthine dehydrogenase Hydroxyacid-oxoacid 0.827 103.040 3-Hydroxybutyric acid* 0.046 0.783 transhydrogenase 0.849 283.068 Xanthosine* <0.001 0.345 Purine metabolism Xanthosine methyltransferase 0.872 135.031 Hypoxanthine* <0.001 1.413 Purine metabolism Xanthine dehydrogenase 0.891 161.045 Glucosan <0.001 0.414 0.927 116.071 Valine* <0.001 1.440 Valine, leucine and isoleucine metabolism Valine dehydrogenase 0.944 101.024 Succinate semialdehyde <0.001 0.390 Alanine, aspartate and glutamate metabolism Aryl-aldehyde dehydrogenase 0.947 346.055 Adenosine monophosphate <0.001 0.320 Purine metabolism (NADP+) 0.965 171.006 Glycerol-2-phosphate <0.001 0.395 Glycerolipid metabolism 0.975 143.034 3-Hexenedioic acid <0.001 0.398 Fatty acid metabolism 0.983 113.024 Ethylmalonic acid* <0.001 0.390 Fatty acid metabolism Acyl-coenzyme A dehydrogenase 1.033 114.056 Proline* 0.001 1.467 Arginine and proline metabolism Proline oxidase 1.036 145.062 * 0.012 1.415 Alanine, aspartate and glutamate metabolism Glutamate synthase 1.098 383.113 S-Adenosylhomocysteine* 0.027 0.779 Cysteine and methionine metabolism 1.352 190.053 N-Acetyl-L-methionine* <0.001 2.296 Cysteine and methionine metabolism Methionine synthase 1.352 121.04 Niacinamide* <0.001 0.633 Nicotinate and nicotinamide metabolism Nicotinamide N-methyltransferase 1.437 130.087 * 0.042 1.184 Valine, and isoleucine metabolism Leucine dehydrogenase 1.689 164.070 * 0.005 1.268 Phenylalanine metabolism Phenylalanine dehydrogenase

1.752 231.134 Spermic acid 2 0.014 1.820

1.766 130.087 Leucine* <0.001 1.432 Valine, leucine and isoleucine metabolism Leucine dehydrogenase 2.056 203.082 * 0.001 1.406 Glycine, serine and threonine metabolism Tryptophan alpha,beta-oxidase 3.936 245.092 N-acetyl-L-tryptophan 0.012 2.961 6.509 134.046 4-Hydroxy-L-threonine <0.001 2.318 7 / 13

16.638 448.304 Glycochenodeoxycholic acid* 0.001 0.354 Bile acid biosynthesis Bile acid-CoA 17.147 313.237 Octadecanedioic acid <0.001 0.534 Beta-ketoacyl -acyl-carrier-protein 18.59 297.242 3-Oxooctadecanoic acid <0.001 1.276 Fatty acid metabolism synthase II 19.123 227.200 FFA C14:0* 0.011 1.366 Fatty acid biosynthesis Acyl-ACP-hydrolase 19.416 277.215 FFA C18:3* 0.005 0.538 Biosynthesis of unsaturated fatty acids Lipoxygenase 19.758 253.216 FFA C16:1 0.032 1.574 Fatty acid biosynthesis Acyl-ACP-hydrolase 19.708 241.216 FFA C15:0 0.004 1.443 Fatty acid metabolism 19.86 279.232 FFA C18:2* 0.015 0.652 Biosynthesis of unsaturated fatty acids Lipoxygenase 19.989 267.232 FFA C17:1 0.004 1.771 Fatty acid metabolism 20.189 509.286 PG C18:1 0.001 0.429 20.396 255.231 FFA C16:0 0.004 1.208 Fatty acid biosynthesis Fatty-acid synthase 20.463 355.263 FFA C24:6 0.004 2.459 Fatty acid metabolism 20.499 305.247 FFA C20:3 <0.001 2.994 Biosynthesis of unsaturated fatty acids Palmitoyl-CoA hydrolase 20.505 608.354 PCPG <0.001 0.388 20.581 281.248 FFA C18:1* 0.001 1.624 Biosynthesis of unsaturated fatty acids Palmitoyl-CoA hydrolase 20.75 571.341 Dolichyl D-xylosyl phosphates 0.041 0.625 20.89 333.278 FFA C22:3 <0.001 2.696 Biosynthesis of unsaturated fatty acids 22.222 283.263 FFA C18:0 0.015 1.123 Fatty acid biosynthesis Acyl-ACP-hydrolase

a Relative metabolite concentrations in HCT samples compared with the corresponding DNT samples. * Validated with standard sample.

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Table S4 The correlation of differential metabolites with clinical information Compounds HBsAg Tumor diameter (cm) AFP (μg/L) ALT (U/L) AST (U/L) (p value, t-test) Positive vs Negative <5 vs >10 <20 vs >200 <60 vs >100 <35 vs >50 2-Octenoylcarnitine 0.006 5'-Methylthioadenosine 0.040 0.039 Arabinosylhypoxanthine 0.046 Chenodeoxyglycocholic acid 0.023 Citramalic acid 0.013 D-fructose 1,6-bisphosphate 0.036 0.035 0.022 FFA(18:2) 0.042 0.041 Gamma-Aminobutyric acid <0.001 Glycerylphosphorylethanolamine 0.026 Glycochenodeoxycholic acid 0.027 Glycocholic acid 0.010 Glycocholic acid fragment 0.024 Glycoursodeoxycholic acid 0.004 Hydroxybutyrylcarnitine 0.008 Hydroxyhexanoycarnitine 0.010 Hypoxanthine 0.002 Isoleucine 0.001 0.010 Glutathione <0.001 Hexanoylcarnitine 0.013 Isoleucyl-L-proline 0.023 9 / 13

Phenylalanine 0.006 Serine <0.001 Threonine 0.003 Tryptophan fragment LysoPC(16:0) 0.027 LysoPC(18:0) 0.022 0.024 LysoPC(18:2) 0.048 LysoPE(18:2) 0.049 N-acetyl-L-tryptophan 0.010 O-Phosphoethanolamine 0.004 0.017 Palmitoylethanolamide S-Adenosylhomocysteine 0.009 0.002 Spermic acid 2 0.041 Succinyladenosine 0.020 Taurine 0.002 Valine 0.038

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Table S5 The changing characteristics of metabolites in serum samples

Cij * Fold change Fold change p value p value Metabolite (Metabolite vs AFP) (HCC/Hepatitis) (HCC/Cirrhosis) (Hepatitis vs HCC) (Cirrhosis vs HCC) Betaine -0.080 0.151 0.124 <0.001 <0.001 Propionylcarnitine 0.033 0.492 0.580 <0.001 0.001 Proline 0.051 0.497 0.718 <0.001 0.006 Methionine 0.132 0.306 0.361 <0.001 <0.001 Isoleucyl-L-proline -0.094 0.814 0.435 0.014 <0.001 Acetylcarnitine 0.142 0.899 0.865 0.261 0.268 3-Hexenedioic acid 0.029 0.966 1.138 0.684 0.198 Carnitine -0.016 0.843 0.986 0.004 0.859 * Cij: Pearson correlation coefficient. 11 / 13

Figure S1. Metabolic profiling of liver tissues. Base peak chromatograms of the distal noncancerous tissue (DNT), adjacent noncancerous tissue (ANT) and hepatocellular carcinoma tissue (HCT) under the ESI+ and ESI- MS mode, respectively. 12 / 13

Figure S2. Study of the metabolic profiles of liver tissues. (a) PCA analysis indicated that the QC samples cluster together. Ions obtained from ESI+ and ESI- modes were merged and used to prepare the figure. R2X is 0.647; (b) The PLS-DA score plot represented a clear classification between cancer and noncancer tissues; (c) S-plot of the PLS-DA mode at the first component. Ions with VIP value>1 were marked with red square. (d) Statistics of the differential metabolites among the three groups. 13 / 13

Figure S3. External validation of betaine and propionylcarnitine in cirrhosis and HCC patients. (a) Relative amount in cirrhosis and HCC patients. (b) Diagnostic accuracy for cirrhosis and HCC by using betaine plus propionylcarnitine. : Cirrhosis; : HCC. ＊, p<0.05; ＊＊, p< 0.01.

Figure S4. Significant differences of citramalic acid in tumor tissue in HBsAg-positive patients from HBsAg-negative ones.