Supplementary material Comprehensive Characterization and Evaluation for Hepatocellular Carcinoma by LC-MS based Serum Metabolomics Journal: Metabolomics Xin Lu 1,3, Huan Nie 1, Yiqun Li 1, Chao Zhan 2, Xia Liu 1, Xiuyun Shi 1,3, Ming Shi 1, Yubao Zhang* 2 and Yu Li* 1,3 1 School of Life Science and Technology, Harbin Institute of Technology, Harbin, China 2 The Affiliated Tumor Hospital, Harbin Medical University, Harbin, China 3 State Key Laboratory of Urban Water Resource and Environment, School of Life Science and Technology, Harbin Institute of Technology, Harbin, China. *Corresponding author: Yu Li, MD/Ph.D., professor Tel/Fax: 86-451-86402691 Email: liyugene@hit.edu.cn ; Address: Room 310, Building 2E, Science Park of Harbin Institute of Technology, No. 2 Yikuang Street, Nangang District, Harbin 150001, China. *Corresponding author: Yubao Zhang, Tel/Fax: 86-451-86298076 Email: zhyb88077@sina.com Address: The Affiliated Tumor Hospital, Harbin Medical University, No. 150, Haping Road, Nangang District, Harbin 150001, China.
Fig. S1 The typical UPLC/Q-TOF MS chromatograms for HCC, HBV and NC group in both positive ion mode (a, b and c for HCC, HBV and NC, respectively) and negative mode (d, e and f for HCC, HBV and NC, respectively)
Fig. S2 Extract ion chromatography, MS spectra and MS/MS spectra of differential metabolites (up-blank) and correspondent standard compounds (down-red) (a) D- Galactose. (b) L-Tryptophan. (c) 3-Indolelactic acid. (d) LyPC(16:0). (e) L-Glutamine. (f) Taurine. (g) Kynurenine (h) Succinic acid
Fig. S3 Box plots for comparing concentration levels of the three discriminative metabolites in different pathological stages and operative conditions. (a) Box plots from normal controls to HCC patients of different stages. (b) Box plots between paired preoperative HCC samples (Pre) and postoperative HCC samples (Post) * p< 0.05 ** p< 0.0001
Table S1 Summary of the differentially expressed metabolites in HCC patients relative to non-hcc control subjects ID Category Marker Database ID Measured (m/z) Retention time(min) Formula Ion mode Trend 1 Sac_1 D-galactose a HMDB00143 179.0561 1.57 C 6 H 12 O 6 ** 2 Sac_2 1-[(5-Amino-5-carboxypentyl)amino]-1- deoxyfructose e 8015298 353.1615 4.96 C 12 H 24 N 2 O 7 M+FA-H ** 3 AA_1 L-Tryptophan a HMDB00929 205.0972 3.21 C 11 H 12 N 2 O 2 4 AA_2 L-Glutamine a HMDB00641 145.0617 1.61 C 5 H 10 N 2 O 3 5 AA_3 L-Phenylalamine a HMDB00159 164.0717 2.98 C 9 H 11 NO 2 6 AA_4 L-Threonine a HMDB00167 118.0508 1.59 C 4 H 9 NO 3 7 AA_5 L-Tyrosine a HMDB00158 180.0652 1.77 C 9 H 11 NO 3 8 AA_7 L-Lysine a HMDB00182 147.1129 1.46 C 6 H 14 N 2 O 2 9 AA_8 L-Serine a HMDB00187 104.0354 1.59 C 3 H 7 NO 3 10 AA_9 L-Leucine a HMDB00687 130.0872 2.75 C 6 H 13 NO 2 11 AA_10 L-Histidine a HMDB00177 156.0755 1.72 C 6 H 9 N 3 O 2 ** 12 AA_11 L-Methionine a HMDB00696 150.0571 1.83 C 5 H 11 NO 2 S ** 13 AA_12 L-Aspartic acid a HMDB00191 132.0305 1.63 C 4 H 7 NO 4 * 14 AA_13 L-Arginine a HMDB00517 173.1039 1.57 C 6 H 14 N 4 O 2 15 AA_14 DL-Kynurenine a HMDB00684 207.0770 2.98 C 10 H 12 N 2 O 3
16 AA_15 L-Glutamate a HMDB00148 146.0460 1.63 C 5 H 9 NO 4 ** 17 DP_1 L-Tryptophyl-L-Phenylalanine a HMDB29090 352.1697 3.89 C 20 H 21 N 3 O 3 18 DP_2 L-(Iso)leucyl-L-Phenylalanine a HMDB13243 279.1700 3.72 C 15 H 22 N 2 O 3 19 DP_3 L-Histidinyl-Cysteine a HMDB28882 149.0246 5.24 C 9 H 14 N 4 O 3 S 20 21 DP_4 DP_5 S-Prenyl-Cysteine a L-Aspartyl-L-Phenylalanine a HMDB12286 HMDB00706 188.0719 279.0987 4.73 3.32 C 8 H 15 NO 2 S C 13 H 16 N 2 O 5 22 DP_6 L-Phenylalanyl-L-Phenylalanine a HMDB13302 311.1404 3.88 C 18 H 20 N 2 O 3 23 DP_7 L-Glycyl-L-Glutamine a HMDB28839 202.0911 4.02 C 7 H 13 N 3 O 4 24 DP_8 L-Glycyl-L-Phenylalanine a HMDB28848 221.0931 3.28 C 11 H 14 N 2 O 3 25 DP_9 L-Glutamyl-L-Glutamate a HMDB28818 273.0804 4.49 C 10 H 14 N 2 O 7 ** 26 OA_1 Succinic acid a HMDB00254 117.0194 1.81 C 4 H 6 O 4 ** 27 OA_2 Citric acid a HMDB00094 191.0198 2.25 C 6 H 8 O 7 ** 28 OA_3 Taurine a HMDB00251 124.0077 1.61 C 2 H 7 NO 3 S ** 29 OA_4 Indoxylsulfuric acid a HMDB00682 212.0025 3.66 C 8 H 7 NO 4 S 30 OA_5 3-Indolelactic acid a HMDB00671 204.0667 4.07 C 10 H 9 O M+FA-H 31 32 ST_1 ST_2 Taurocholate a Taurochenodeoxycholate a HMDB00036 HMDB00951 514.2837 498.2889 4.73 5.16 C 26 H 45 NO 7 S C 26 H 45 NO 6 S ** ** 33 ST_3 Glycocholate a HMDB00138 466.3159 4.97 C 26 H 43 NO 6 * 34 ST_4 Glycochenodeoxycholate a HMDB00637 448.3075 5.61 C 32 H 51 NO 11 * 35 ST_5 3α,7α-Dihydroxy-5beta-cholestan-26-al b C05445 463.3063 6.58 C 26 H 42 O 4 M+FA-H *
36 ST_6 3-β-Hydroxyandrost-5-en-17-one sulfate c 57905 367.1599 5.07 C 19 H 28 O 5 S 37 ST_7 7α-Hydroxycholest-4-en-3-one c 17899 445.3326 11.64 C 27 H 44 O 2 M+FA-H 38 ST_8 Chenodeoxycholate c 36234 391.2853 6.90 C 24 H 40 O 4 39 LyPC_1 LysoPC(16:0) a HMDB10382 496.3410 8.00 C 24 H 50 NO 7 P 40 41 42 43 LyPC_2 LyPC_3 LyPC_4 LyPC_5 LysoPC(18:0) a LysoPC(18:1(11Z)) a LysoPC(20:3(8Z,11Z,14Z)) a LysoPC(18:2(9Z,12Z)) a HMDB10384 HMDB10385 HMDB10394 HMDB10386 524.3728 522.3568 546.3544 564.3321 9.99 8.76 8.25 7.44 C 26 H 54 NO 7 P C 26 H 52 NO 7 P C 28 H 52 NO 7 P C 26 H 50 NO 7 P M+FA-H 44 LyPC_6 LysoPC(20:4(8Z,11Z,14Z,17Z) a HMDB10396 588.3315 7.69 C 28 H 50 NO 7 P M+FA-H 45 LyPC_7 LysoPC(17:0) a HMDB12108 554.3473 9.40 C 25 H 52 NO 7 P M+FA-H 46 LyPC_8 LysoPC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)) a HMDB10404 612.3308 7.62 C 30 H 50 NO 7 P M+FA-H 47 LyPC_9 LysoPC(P-16:0) a HMDB10407 524.3364 8.94 C 24 H 50 NO 6 P M+FA-H 48 LyPC_10 LysoPC(18:2(9Z,12Z)) a HMDB10386 564.3325 7.71 C 26 H 50 NO 7 P M+FA-H 49 50 51 52 53 54 55 LyPE_1 LyPE_2 LyPE_3 PC_1 PC_2 PC_3 PC_4 LysoPE(18:2(9Z,12Z)/0:0) a LysoPE(0:0/18:0) a LysoPE(20:2(11Z,14Z)/0:0) a 1-Linoleoyl glycerophosphochline a PC(O-18:2(9Z,12Z)/12:0) d PC(15:0/0:0) d PC(P-18:0/0:0) d HMDB11507 HMDB11129 HMDB11513 HMDB61692 40020 40282 40405 478.2934 480.3104 504.3099 520.3409 548.3716 482.3241 508.3769 7.60 10.16 7.72 7.63 9.23 7.50 9.20 C 23 H 44 NO 7 P C 23 H 48 NO 7 P C 25 H 48 NO 7 P C 26 H 50 NO 7 P C 28 H 54 NO 7 P C 23 H 48 NO 7 P C 26 H 54 NO 6 P *
56 PC_5 PC(16:0) d 40341 540.3325 8.44 C 24 H 50 NO 7 P M+FA-H 57 PC_6 PC(O-16:0/2:0) d 40075 568.3640 10.19 C 26 H 54 NO 7 P M+FA-H 58 PC_7 PC(16:1(9Z)/2:0) d 39434 580.3255 5.47 C 26 H 50 NO 8 P M+FA-H 59 PE_1 PE(P-16:0/0:0) d 46719 436.2838 8.89 C 21 H 44 NO 6 P 60 PE_2 PE(P-18:0/0:0) d 46720 464.3158 10.53 C 23 H 48 NO 6 P 61 PE_3 PE(20:0/20:4(5Z,8Z,11Z,14Z)) d 60633 840.5761 8.95 C 45 H 82 NO 8 P M+FA-H 62 PG_1 PG(P-16:0/15:1(9Z)) d 79910 357.2414 10.31 C 37 H 71 O 9 P M+Na+H ** 63 FA_1 (10E)-9-Oxo-10-hexadecenoic acid e 27023196 269.2108 6.91 C 16 H 28 O 3 ** 64 FA_2 12-HEPE a HMDB10202 319.2266 9.45 C 20 H 30 O 3 65 FA_3 EPA c 400199 301.2176 8.97 C 20 H 30 O 2 66 FA_4 Docosahexaenoic acid a HMDB61088 327.2336 10.81 C 22 H 32 O 2 * 67 FA_5 13(S)-HODE d 45667 295.2283 8.04 C 18 H 32 O 3 68 FA_6 (±)5-HETE c 28209 319.2286 8.96 C 20 H 32 O 3 69 FA_7 6-trans-LTB4 a HMDB05087 335.3845 6.46 C 20 H 32 O 4 70 FA_8 (±)4-HDoHE c 72624 343.228 9.14 C 22 H 32 O 3 71 FA_9 Arachidonic Acid c 15843 303.2334 10.98 C 20 H 32 O 2 * 72 FA_10 9-Octadecenoic acid a HMDB02066 345.2440 10.03 C 18 H 33 FO 2 M+FA-H 73 FA_11 (R)-3-Hydroxy-hexadecanoic acid a HMDB10734 271.2276 7.52 C 16 H 32 O 3 ** 74 C4 Butyryl-L-carnitine c 21949 232.1537 3.11 C 11 H 21 NO 4 75 C8 Octanoyl-L-carnitine c 18102 288.2167 4.56 C 15 H 29 NO 4
76 C8:1 Octenoyl-L-carnitine c 71012 286.2012 4.25 C 15 H 27 NO 4 ** 77 C9 Nonanoyl-L-carnitine c 70997 302.2306 4.71 C 16 H 31 NO 4 78 C10 Decanoyl-L-carnitine c 28717 316.2482 5.12 C 17 H 33 NO 4 79 C11 Undecanoyl-L-carnitine a HMDB13321 330.2631 5.27 C 18 H 35 NO 4 80 C12 Dodecanoyl-L-carnitine c 77086 344.2786 5.81 C 19 H 37 NO 4 * 81 C12:1 Dodecenoyl-L-carnitine a HMDB13326 342.2652 5.40 C 19 H 35 NO 4 * 82 C14 Tetradecanoyl-L-carnitine a HMDB05066 372.3108 6.91 C 21 H 41 NO 4 * 83 C14:1 Tetradecenoyl-L-carnitine a HMDB13329 370.2958 6.25 C 21 H 39 NO 4 * 84 C16 Palmitoyl-L-carnitine a HMDB00222 400.3433 8.28 C 23 H 45 NO 4 * 85 C16:1 Hexadecenoyl-L-carnitine a HMDB13207 398.3280 7.29 C 23 H 43 NO 4 ** 86 C18 Stearoyl-L-carnitine a HMDB00848 428.3739 9.61 C 25 H 49 NO 4 ** 87 C18:1 Octadecenyl-L-carnitine a HMDB05065 426.3588 8.58 C 25 H 47 NO 4 ** 88 C18:2 Octadecadienyl-L-carnitine a HMDB06469 424.3429 7.66 C 25 H 45 NO 4 ** 89 NA_1 Uridine a HMDB00269 289.0716 5.31 C 9 H 12 N 2 O 6 M+FA-H ** * p< 0.05; ** p< 0.0001, up-regulated in HCC patients; down-regulated in HCC patients Referred databases: a. HMDB, b. KEGG, c. ChEBI, d. METLIN, e. ChemSpider