Human Metabolome

Human Metabolome Technologies Inc. (HMT) is a leading metabolomics service provider company established on July 2003 based on capillary electrophoresis mass spectrometry (CE-MS) technologies. Technologies The company was listed on the Mothers section of Tokyo Stock Exchange in December 2013. Our main Commissioned metabolome analysis services business is commissioned metabolomics analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS): We have a time-tested track record in a number of different fields including medical sciences, pharmaceuticals, food products, fermentation, and cosmetics. With an aim of contributing in a wide range of fields, we will now set our sights on untapped areas such as the environment, energy, and chemical industries.

History

Jul 2003 Founded in Suehiromachi in Tsuruoka, Yamagata Prefecture with capital of 10 million yen.

Jun 2004 Concluded a joint research agreement with Ajinomoto Co., Inc.

May 2009 Commenced the “HMT Research Grant for Young Leaders”

Aug 2012 Launched a cancer research specialized package, “C-SCOPE.”

Oct 2012 Established a sales subsidiary, “Human Metabolome Technologies America, Inc.” in Massachusetts, USA

Sep 2013 Registered the patent “The biomarker for depression, the measuring method for the biomarker of depression, and the program and storage for the diagnostic method” (patent number 5372213) in Japan

Dec 2013 Listed on the Mothers section of the Tokyo Stock Exchange

Jan 2016 Established a biomarker business company “HMT Biomedical Co., Ltd.” in Yokohama, Kanagawa, Japan

May 2017 Established a sales subsidiary, “Human Metabolome Technologies Europe B.V.” in Leiden, Netherlands

Apr 2018 Launched functional lipidomics specialized package “Mediator Scan”

Human Metabolome Technologies Inc.

■Headquarters and Laboratory ■Human Metabolome Technologies America, Inc. 246-2 Mizukami, Kakuganji, Tsuruoka, Yamagata 24 Denby Road, Suite217 Boston, MA 02134 997-0052, Japan Phone： +1-617-871-9940

■Tokyo Office ■Human Metabolome Technologies Europe B.V. Stern Chuo Bldg. 5F, 2-9-6 Shinkawa, Chuo, Tokyo J.H. Oortweg 21, 2333 CH Leiden, Netherlands 104-0033, Japan Phone: +31-71-332-2040 Phone: +81-3-3551-2180 Fax: +81-3-3551-2181 ■HMT BioMedical Inc. 2-11-5 Shin-Yokohama, Kohoku-ku, Yokohama 222-0033 Phone: +81-45-534-9316 Fax: +81-45-534-9317

Dealer

201810ver.4 What is Metabolomics? Application of Metabolomics

Inside the body of living organisms including humans, For instance, in sickness, dietary, or environmental changes, metabolites in the living organisms (i.e., in the there exist small molecules such as amino acids, blood, urine, tissue, etc.) also change accordingly. Observation of these changes is useful for discovery of sugars, and organic acids. Most of these substances biomarkers, metabolites showing disease-specific rise and fall: we can use them to diagnose diseases, and are “metabolites” produced through enzymatic activi- also for revealing the elucidation of action mechanisms of medications, and evaluation of phenotypic effects ties. The methodology to analyze molecular species or by genetic mutations and abnormal protein function on metabolism. In the areas of APIs, fermentation, and concentrations of many metabolites at once is either production of food materials, metabolomics is expected to complement insufficiency of data pertaining to called “metabolome analysis” or “metabolomics.” product quality which gene- or protein-based assessment do not go far enough to provide. This is because Metabolites interact with each other through “metabo- metabolomics visualizes comprehensive status of low-molecular-weight compounds in samples. lism,” chemical reaction networks in living organisms. As seen in the medical field, a variety of fields including the agricultural, dietetics, environmental sciences, Metabolites produced by certain substances also as well as the chemical industries, have also found metabolomics to be a very useful tool. Examples of uses serve as precursors for subsequent reactions. are: screening of substances in agricultural and Comprehensive profiling of metabolites helps us moni- livestock products, optimization of growth condi- tor dynamics of the metabolism, and provides us with tions, evaluations in selective breeding, investigation an even better understanding of the life phenomenon. of correlations between savor and components and discovery of novel functional compounds. The range of fields to which metabolomics can be applied continues to grow. HMT is well positioned to utilize its wealth of experience in metabolome analysis in What is CE-MS? order to contribute to breakthroughs in R&D in a variety of fields.

There are thought to be at least 3,000 species of metabolites between 50 to 1,000 Daltons of Metabolome Analysis molecular mass, so individual analysis of each metabolite is too time-consuming to cover the Medicine entire metabolism. Therefore, it is necessary to employ methods to measure a number of com- pounds precisely and accurately with high sensitivity at once. It is also important to select an Pathological mechanism elucidation appropriate method based on intrinsic chemical prop- Discovery of erties of metabolites such as solubility and ionicity, etc. CE-MS Water-soluble ionic substances Material etiologic factors Agricultural Production Cosmetics science Capillary Electrophoresis (CE) is a suitable method for separation of water-soluble metabolites that exist in Nucleic acids Sugar phosphates Phosphate substances Optimization of Safety assessment Selective breeding production systems Improvement of productivity Carbohydrate derived Composition effects large numbers in living organisms. The tandem connec- metabolites Organic acids Amino acids Functional assessment Ecological investigations mechanistic studies tion of CE and Mass Spectrometry (a combined capillary Amino acid byproducts Polyamines Chemistry Pharmaceuticals electrophoresis system and mass spectrometer, referred Biofuels Effects and efficacy to as “CE-MS”), which is capable of detecting tiny Biorefineries Toxicity and safety LC-MS Lipid-soluble neutral substances Companion diagnostic amount of substances, the HMT’s base technology, Food products Diagnostics markers (CDx) enables high sensitivity analysis of up to several hundred Food analysis Diagnostics development different metabolites. Based on this unique method, Lipids Fatty acids Acylcarnitines Functional food evaluation Therapeutic effects HMT conducts analysis of metabolites and develops Discovery of functional assessment Bile acids Polyphenols Steroids components new technologies. Employing CE-MS in conjunction with LC-MS based on liquid chromatography (LC) also covers a more diverse range of chemical properties.

02 HMT HMT 03 Focus further on metabolism Analysis Analysis Cost performance model targets using labeling analysis targets CE-TOFMS CE-TOFMS Roughly Quantitation 30+24 Quantitation (with units e.g. μM) (with units e.g. μM) 900 substances substances Basic Scan Relative quantitation Relative quantitation (Without units) (Without units) (Peak area value) (Peak area value)

Ideal for monitoring basic metabolic changes, and analyzing mechanisms. Stable isotope labeling analysis shows the process in which labeled compounds are metabolized. Makes it possible to obtain metabolic information that serves as Ideal for deeper analysis regarding the central energy metabolism and the base for advancing research a step further. amino acid metabolism.

Specifications...Time for delivery: 60 days / Analysis device: CE-TOFMS / Reports: Relative values of detected substances, Specifications...Time for delivery: 60 days / Analysis device: CE-TOFMS / Reports: Relative values of each isotopomer of group comparisons using detection data, principal component analysis, heat map, pathway map / Analysis targets: Roughly detected substances, group comparisons using detection data, pathway map / Analysis targets: 30 substances (Anion: 900 substances appearing on CE-MS annotation list (p. 10-12) / Targets (examples): Amino acids, , nucleosides, glycolysis, tricarboxylic acid (TCA) cycle, pentose phosphate pathway, ATP, ADP, AMP, etc.), 24 substances (Cation: Amino nucleobase, Coenzyme A (CoA) derivatives, organic acids, nicotinamide coenzymes, etc. acid, citrulline, ornithine, glutathione) Optional services...Q-OPTION 110, Q-OPTION 400, Peptide Search * Labeled compounds to be added or administered must be prepared by customers.

Analysis targets Example of report LC-TOFMS Roughly Analysis 〈Information on detected compounds〉 Integration of advanced technologies targets 300 substances CE-TOFMS HMT DB † Relative Area Comparative Analysis ID 0h 6h 12h 18h 6h vs 0h 12h vs 0h 18h vs 0h Roughly Compound name ¶ || ¶ || ¶ || Quantitation Mean S.D. Mean S.D. Mean S.D. Mean S.D. Ratio p-value Ratio p-value Ratio p-value 900 (with units e.g. μM) A_0003 Pyruvic acid 3.6E-04 6.8E-05 2.8E-04 5.7E-05 2.3E-04 4.6E-05 2.6E-04 N.A. 0.8 0.109 0.7 0.073 0.7 N.A. substances A_0004 Butyric acid 7.7E-05 6.2E-05 4.7E-05 9.7E-06 9.6E-05 4.4E-05 1.1E-04 5.1E-05 0.6 0.343 1.2 0.600 1.4 0.389 Dual Scan Relative quantitation (Without units) A_0005 Lactic acid 1.8E-01 3.1E-02 1.5E-01 1.1E-02 8.3E-02 2.6E-02 9.4E-02 1.8E-02 0.8 0.091 0.5 8.4E-04 *** 0.5 0.002 ** (Peak area value) A_0006 Valeric acid 8.5E-05 N.A. N.D. N.A. 1.0E-04 1.9E-05 1.2E-04 1.8E-05 <1 N.A. 1.2 N.A. 1.4 N.A. A_0007 3-Hydroxybutyric acid 3.0E-03 1.4E-03 4.0E-03 1.4E-03 1.4E-02 3.1E-03 2.4E-02 5.7E-03 1.3 0.294 4.5 6.7E-04 *** 7.8 8.9E-04 *** A_0008 2-Hydroxybutyric acid 6.5E-04 1.8E-04 4.2E-04 1.4E-04 1.1E-03 2.1E-04 1.7E-03 5.6E-04 0.6 0.061 1.7 0.006 ** 2.7 0.009 ** Adds LC-MS analysis to Basic Scan, ideal for energy metabolism screening, A_0009 2-Hydroxyisobutyric acid 1.2E-04 2.1E-05 1.2E-04 2.1E-05 9.3E-05 3.7E-05 1.2E-04 3.0E-05 1.1 0.678 0.8 0.313 1.0 0.837 A_0010 Glyceric acid 6.2E-05 1.1E-05 5.5E-05 6.6E-06 4.2E-05 2.6E-06 3.9E-05 6.0E-06 0.9 0.251 0.7 0.011 * 0.6 0.005 ** including lipid metabolism. Optimal for monitoring basic metabolic changes, A_0011 Fumaric acid 4.2E-03 1.6E-03 3.8E-03 8.8E-04 2.1E-03 3.8E-04 2.4E-03 3.1E-04 0.9 0.662 0.5 0.038 * 0.6 0.060 and analyzing mechanisms. A_0012 4-Oxovaleric acid N.D. N.A. N.D. N.A. 5.6E-05 8.2E-06 7.2E-05 1.5E-05 N.A. N.A. 1< N.A. 1< N.A.

Specifications...Time for delivery: 60 days / Analysis device: CE-TOFMS and LC-TOFMS / Reports: Relative values of detected substances, Group comparisons using detection data, principal component analysis, heat map, pathway map / Analysis 〈Heat map〉 〈Pathway map〉 targets: Roughly 900 substances appearing on CE-MS annotation lists (p. 10-12), roughly 300 substances appearing on LC-MS annotation lists (p13)/ Representative targets: Amino acids, types of , nucleosides, nucleobase, CoA derivatives, organic acids, nicotinamide coenzymes, fatty acids, acyl carnitine, bile aid, polyphenol, steroids, etc. Optional services...Q-OPTION 110, Q-OPTION 400, Peptide Search * LC-TOFMS analysis can also be ordered as a separate service:. Please contact us for details.

Biomarker discovery model, Analysis reaching stages of astonishment targets CE-TOFMS Roughly Quantitation 900 (with units e.g. μM) substances 〈Principal component analysis〉 Advanced Scan Relative quantitation (Without units) (Peak area value)

Ideal approach for screening to understand metabolic changes occurring over a broad range, along with discovery of unknown biomarkers and functional components.

Specifications...Time for delivery: 60 days / Analysis device: CE-TOFMS / Reports: Relative values of detected substances, group comparisons using detection data, principal component analysis, heat map, pathway map / Analysis targets: Roughly 900 substances appearing on CE-MS annotation lists (p. 10-12), peptide libraries, HMDB libraries, libraries, unknown peaks outside of these libraries Optional services...Q-OPTION 110, Q-OPTION 400

04 HMT HMT 05 Targeted analysis model that continues to evolve

High-sensitivity, high-accuracy targeted analysis plan Example of report

focusing on the central energy metabolic pathways 〈Information on detected compounds (quantification using three-point calibration)〉

HMT DB Concentration (nmol/g) Comparative Analysis ID 0h 3h 18h 0h 3h 18h 3h vs 0h 18h vs 0h Compound name L0-1 L0-2 L0-3 L0-4 L0-5 L3-1 L3-2 L3-3 L3-4 L3-5 L18-1 L18-2 L18-3 L18-4 L18-5 Mean S.D. Mean S.D. Mean S.D. Ratio ¶ p-value || Ratio ¶ p-value || A_0001 NAD+ 484 479 401 487 547 491 646 466 567 544 191 222 318 239 265 480 51.7 543 70.7 247 47.6 1.1 0.149 0.5 7.9E-05 *** A_0002 cAMP 0.54 0.71 0.47 0.58 0.65 0.68 0.74 0.66 0.85 0.74 0.39 0.47 0.64 0.47 0.43 0.59 0.09 0.73 0.07 0.48 0.10 1.2 0.027 * 0.8 0.106 A_0003 cGMP 0.03 0.04 N.D. N.D. 0.05 0.03 0.02 0.02 0.03 0.03 0.01 0.02 0.02 0.02 0.01 0.04 0.01 0.03 0.01 0.02 0.00 0.7 0.229 0.5 0.073 By carefully selecting 116 targets related to glycolysis, tricarboxylic acid (TCA) cycle, pentose phosphate pathway, amino A_0004 136 101 99.5 119 162 157 206 118 179 160 113 109 104 97.8 94.7 124 26.3 164 32.5 104 7.63 1.3 0.065 0.8 0.169 A_0005 NADH 2.72 3.14 2.37 2.76 3.45 2.76 3.14 2.63 3.00 2.73 1.96 2.13 3.15 2.65 2.01 2.89 0.42 2.85 0.21 2.38 0.51 1.0 0.863 0.8 0.124 acid metabolism, and , C-SCOPE achieved the highest level of sensitivity and quantitative range A_0006 ADP-ribose 252 234 169 206 255 272 283 195 222 271 67.6 99.3 72.0 76.3 152 223 36.0 249 38.1 93.4 34.8 1.1 0.312 0.4 4.1E-04 *** A_0007 Mevalonic acid N.D. N.D. N.D. N.D. N.D. N.D. 0.08 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.A. 0.08 N.A. N.D. N.A. 1< N.A. N.A. N.A. A_0008 UDP-glucose 340 380 286 384 299 348 480 393 272 359 49.1 61.5 103 94.8 71.8 337 45.0 370 75.5 76.1 22.6 1.1 0.431 0.2 2.8E-05 *** in the world. We calculate metabolic parameters from absolute quantitative values, and draw out rational interpretations. A_0009 21.0 8.26 18.2 18.1 28.2 12.7 30.5 15.4 26.1 27.3 4.25 8.17 7.23 4.85 6.11 18.7 7.15 22.4 7.85 6.12 1.63 1.2 0.461 0.3 0.015 * A_0010 NADP+ 127 154 125 142 158 146 219 139 174 184 72.0 99.6 162 90.8 135 141 15.0 172 32.2 112 36.1 1.2 0.101 0.8 0.148 Specifications...Time for delivery: 60 days / Subject specimen: Animal tissue, cultured cells, blood, culture medium, microorganisms (yeast and coliform only) / Analysis device: CE-TOFMS and CE-QqQ MS / Reports: Absolute quantitative values of detected substances, group comparisons using detection data, principal component analysis, heat map, pathway map, metabolic parameters / Analysis targets: 116 metabolites 〈Pathway map〉 Optional services...LC-MS analysis 〈30 metabolic parameters〉 List of C-SCOPE analysis targets (116 substances)

2, 3-BPG Erythrose 4-phosphate Methionine 2-Hydroxyglutaric acid Folic acid (total) Mevalonic acid 2-Oxoglutaric acid Fructose 1,6-diphosphate N,N-Dimethylglycine 2-Oxoisovaleric acid Fructose 1-phosphate N-Acetylglutamate 2-Phosphoglyceric acid Fructose 6-phosphate NAD+ 3-Phosphoglyceric acid Fumarate NADH 6-Phosphogluconic acid GABA NADP+ Acetoacetyl-CoA Galactose 1-phosphate NADPH Acetyl-CoA GDP Ornithine Adenine Glucose 1-phosphate Phenylalanine Adenosine Glucose 6-phosphate Phosphocreatine Adenylosuccinic acid Glutamate Phosphoenolpyruvate ADP Glutamine PRPP ADP-ribose Glutathione (ox) Proline Alanine Glutathione (red) Putrescine AMP G3P Pyruvic acid Arginine Glycerol 3-phosphate Ribose 1-phosphate Argininosuccinic acid Glycine Ribose 5-phosphate Asparagine Glycolate Ribulose 5-phosphate Aspartic acid Glyoxylate S-Adenosylhomocysteine ATP GMP S-Adenosylmethionine Betaine GTP Sarcosine Betaine aldehyde Guanine S7P cAMP Guanosine Serine Carbamoyl aspartic acid Histidine Spermidine HMG-CoA Spermine Carnitine Homocysteine Succinic acid Carnosine Homoserine Threonine cGMP Hydroxyproline Tryptophan Choline Tyrosine cis-Aconitic acid IMP UDP-glucose Citrate Inosine Urea Citrulline Isocitrate Uric acid CoA Isoleucine Valine Creatine Lactic acid Xanthine Creatinine Leucine XMP Cystathionine Lysine Xylulose 5-phosphate Cysteine Malic acid β-Alanine Figure 12. Adenylate energy charge and total adenylate DHAP Malonyl-CoA

06 HMT HMT 07 Scan lipid mediators

Functional lipidomics specialized package focusing on lipid mediators involved in signal transmission

Comprehensive analysis of lipidomics about 400 species of lipid-soluble metabolites, including oxylipins * Target substances are not the same as List of analysis targets (400 substances) those on the LC-MS annotation list (p.13) and lysophospholipids which are typical lipid mediators. Optimal for mechanism elucidation and Thyroxine Fatty acid LysoPAF Lysophosphatidylglycerol Glucosylceramide(d18:1/22:2) biomarker discovery for a wide range of diseases for which involvement of lipid mediators is suspected, Triiodothyronine FA(19:0) Glucosylceramide(d18:1/24:0) FA(10:0) LysoPAF(16:0) LysoPAF(18:0) Ursodeoxycholic acid LPG(12:0) LPG(20:0) such as immunity, inflammation, allergic disorders, diabetes, arteriosclerosis, cancer, and dementia. FA(11:0) FA(19:1) LPG(14:0) LPG(20:1) Glucosylceramide(d18:1/24:1) Arachidonic acid metabolites Lysophosphatidylcholine FA(11:1) FA(19:2) LPG(15:0) LPG(20:2) Glucosylceramide(d18:1/24:2) 5-HETE FA(12:0) FA(20:0) LPC(12:0) LPC(20:0) LPG(16:0) LPG(20:3) Lactosylceramide Specifications...Time for delivery: 90 days / Subject specimen: Blood (plasma, serum) (expansion of applicable sample types is 5-oxoETE FA(12:1) FA(20:1) LPC(14:0) LPC(20:1) LPG(16:1) LPG(20:4) 5,6-diHETE Lactosylceramide(d18:1/14:0) planned.) / Analysis device: LC-QTrap / Reports: Relative values of detected substances, group comparisons using detection FA(13:0) FA(20:2) LPC(15:0) LPC(20:2) LPG(17:0) LPG(20:5) 5,15-diHETE Lactosylceramide(d18:1/16:0) FA(13:1) FA(20:3) LPC(16:0) LPC(20:3) LPG(18:0) LPG(22:0) data, principal component analysis, heat map, pathway map / Analysis targets: 400 substances (may be changed in the future) 6-keto Prostaglandin F1α Lactosylceramide(d18:1/16:1) FA(14:0) FA(20:4) LPC(16:1) LPC(20:4) LPG(18:1) LPG(22:1) 8,15-diHETE Lactosylceramide(d18:1/18:0) / Main targets (examples): Fatty acids, acylcarnitine, lysophospholipids (LPA, LPC, LPE, LPG, LPI, LPS), eicosanoids, EPA and FA(14:1) FA(20:5) LPC(17:0) LPC(20:5) LPG(18:2) LPG(22:2) 12-HETE Lactosylceramide(d18:1/18:1) FA(15:0) FA(21:0) LPC(18:0) LPC(22:0) LPG(18:3) LPG(22:3) DHA metabolites, ceramide, endogenous cannabinoid, etc. 15-deoxy Prostaglandin J2 Lactosylceramide(d18:1/20:0) FA(15:1) FA(21:1) LPC(18:1) LPC(22:1) LPG(19:0) LPG(22:4) Combination with other service packages such as Advanced Scan and Basic Scan provides further comprehensive analysis. 15-HETE Lactosylceramide(d18:1/20:1) FA(16:0) FA(22:0) LPC(18:2) LPC(22:2) LPG(22:5) 15-oxoETE Lactosylceramide(d18:1/22:0) FA(16:1) FA(22:1) LPC(18:3) LPC(22:3) LPG(22:6) 20-hydroxy Leukotriene B4 Lactosylceramide(d18:1/22:1) FA(16:2) FA(22:2) LPC(19:0) LPC(22:4) LPG(24:0) Leukotriene B4 Lactosylceramide(d18:1/22:2) FA(17:0) FA(22:3) LPC(22:5) LPG(24:1) Lipoxin A4 Lactosylceramide(d18:1/24:0) FA(17:1) FA(22:4) LPC(22:6) Example of report Lipoxin B4 Lysophosphatidic acid Lactosylceramide(d18:1/24:1) FA(18:0) FA(22:5) LPC(24:0) Prostaglandin D2 LPA(12:0) LPA(20:0) Lactosylceramide(d18:1/24:2) FA(18:1) FA(22:6) LPC(24:1) Prostaglandin F2α LPA(14:0) LPA(20:1) Ceramide FA(18:2) FA(23:0) Lysophosphatidylethanolamine Prostaglandin H2 LPA(15:0) LPA(20:2) Ceramide-1P(d18:1/14:0) FA(18:3) FA(23:1) LPE(12:0) LPE(20:0) 〈Pathway map〉 Prostaglandin I2 LPA(16:0) LPA(20:3) Ceramide-1P(d18:1/16:0) FA(24:0) LPE(14:0) LPE(20:1) Thromboxane B2 LPA(16:1) LPA(20:4) Ceramide-1P(d18:1/16:1) FA(24:1) LPE(15:0) LPE(20:2) EPA metabolites LPA(17:0) LPA(20:5) Ceramide-1P(d18:1/18:0) Acylcarnitine LPE(16:0) LPE(20:3) LPA(18:0) LPA(22:0) 5-HEPE Ceramide-1P(d18:1/18:1) AC(10:0) AC(19:0) LPE(16:1) LPE(20:4) LPA(18:1) LPA(22:1) 12-HEPE Ceramide-1P(d18:1/20:0) AC(11:0) AC(19:1) LPE(17:0) LPE(20:5) LPA(18:2) LPA(22:2) 15-HEPE Ceramide-1P(d18:1/20:1) AC(11:1) AC(20:0) LPE(18:0) LPE(22:0) LPA(18:3) LPA(22:3) 18-HEPE Ceramide-1P(d18:1/22:0) AC(12:0) AC(20:1) LPE(18:1) LPE(22:1) LPA(19:0) LPA(22:4) Lipoxin A5 Ceramide-1P(d18:1/22:1) AC(12:1) AC(20:2) LPE(18:2) LPE(22:2) LPA(22:5) Prostaglandin D3 Ceramide-1P(d18:1/22:2) AC(13:0) AC(20:3) LPE(18:3) LPE(22:3) LPA(22:6) Prostaglandin E3 Ceramide-1P(d18:1/24:0) AC(13:1) AC(20:4) LPE(19:0) LPE(22:4) LPA(24:0) Resolvin E1 Ceramide-1P(d18:1/24:1) LPE(22:5) AC(14:0) AC(20:5) LPA(24:1) Ceramide-1P(d18:1/24:2) DHA metabolites AC(14:1) AC(22:0) LPE(22:6) Sphinganine 4-HDoHE LPE(24:0) Ganglioside AC(15:0) AC(22:1) Sphinganine(d16:0) 7-HDoHE LPE(24:1) GD1(d18:1/16:0) AC(15:1) AC(22:2) Sphinganine(d18:0) 7,17-diHDoHE GD1(d18:1/18:0) AC(16:0) AC(22:3) Lysophosphatidylinositol Sphinganine(d20:0) 10-HDoHE GD3(d18:1/16:0) AC(16:1) AC(22:4) LPI(12:0) LPI(20:0) Sphinganine(d22:0) 13-HDoHE GD3(d18:1/18:0) AC(17:0) AC(22:5) LPI(14:0) LPI(20:1) Sphinganine(d24:0) 14-HDoHE GM3(d18:1/12:0) AC(17:1) AC(22:6) LPI(15:0) LPI(20:2) 17-HDoHE Sphingosine GM3(d18:1/12:1) AC(18:0) AC(24:0) LPI(16:0) LPI(20:3) 20-HDoHE Sphingosine(d16:1) GM3(d18:1/14:0) AC(18:1) AC(24:1) LPI(16:1) LPI(20:4) Protectin DX Sphingosine(d18:1) GM3(d18:1/14:1) AC(18:2) LPI(17:0) LPI(20:5) Resolvin D1 Sphingosine(d20:1) GM3(d18:1/16:0) AC(18:3) LPI(18:0) LPI(22:0) Resolvin D2 Sphingosine(d22:1) GM3(d18:1/16:1) Endocannabionoid LPI(18:1) LPI(22:1) Sphingosine(d24:1) GM3(d18:1/18:0) Steroid LPI(18:2) LPI(22:2) Acylethanolamine(16:0) GM3(d18:1/18:1) 7-Dehydrocholesterol LPI(18:3) LPI(22:3) Sphinganine-1P Acylethanolamine(18:0) GT1(d18:1/16:0) 17-Hydroxyprogesterone LPI(19:0) LPI(22:4) Sphinganine-1P(d16:0) Acylethanolamine(18:1) GT1(d18:1/18:0) Acylethanolamine(18:2) 20-Hydroxycholesterol LPI(22:5) Sphinganine-1P(d18:0) Acylethanolamine(18:3) 22-Hydroxycholesterol LPI(22:6) Sphinganine-1P(d20:0) Acylethanolamine(20:3) Aldosterone LPI(24:0) Sphinganine-1P(d22:0) Acylethanolamine(20:4) Chenodeoxycholic acid LPI(24:1) Sphinganine-1P(d24:0) Acylethanolamine(20:5) Cholic acid Lysophosphatidylserine Sphingosine-1P Corticosterone Acylethanolamine(22:4) LPS(12:0) LPS(20:0) Sphingosine-1P(d16:1) Cortisol Acylethanolamine(22:6) LPS(14:0) LPS(20:1) Sphingosine-1P(d18:1) Cortisone 2-Arachidonoylglycerol LPS(15:0) LPS(20:2) Sphingosine-1P(d20:1) Deoxycholic acid Platelet-activating factor (PAF) LPS(16:0) LPS(20:3) Sphingosine-1P(d22:1) Desmosterol LPS(16:1) LPS(20:4) Sphingosine-1P(d24:1) PAF(14:0) PAF(19:0) DHEA-S LPS(17:0) LPS(20:5) Glucosylceramide PAF(15:0) PAF(20:4) Estradiol LPS(18:0) LPS(22:0) PAF(16:0) PAF(20:5) Estriol Glucosylceramide(d18:1/14:0) LPS(18:1) LPS(22:1) PAF(16:1) PAF(20:6) Estrone Glucosylceramide(d18:1/16:0) LPS(18:2) LPS(22:2) PAF(16:2) PAF(22:4) Lanosterol Glucosylceramide(d18:1/16:1) LPS(18:3) LPS(22:3) PAF(17:0) PAF(22:5) Lithocholic acid Glucosylceramide(d18:1/18:0) LPS(19:0) LPS(22:4) PAF(18:0) PAF(22:6) Pregnenolone Glucosylceramide(d18:1/18:1) LPS(22:5) PAF(18:1) Progesterone Glucosylceramide(d18:1/20:0) LPS(22:6) PAF(18:2) Squalene Glucosylceramide(d18:1/20:1) LPS(24:0) PAF(18:3) Testosterone Glucosylceramide(d18:1/22:0) LPS(24:1) Glucosylceramide(d18:1/22:1)

08 HMT HMT 09 Q110 :Target compounds covered by both "Q-OPTION Premium 400" and "Q-OPTION Standard 110" CE-MS Annotation List Q:Target compounds covered by only "Q-OPTION Premium 400" Grayed-out compounds are poor in linearity and recovery rate, so the results are for reference purpose only. Glucaric acid 10-Hydroxydecanoic acid Lipoamide Q Mevalonic acid Q110 Gluconic acid 6-Hydroxydopamine Lumazine Mimosine A Q Aminoacetone Q110 Carnosine Q N1, N12-Diethylspermine Gluconolactone Q 2-Hydroxyglutaric acid Q110 Lys Minocycline Q 4-Acetamidobutanoic acid Q 2-Aminoadipic acid cCMP7 Q Digalacturonic acid Glucosamine 6-Hydroxyhexanoic acid Lysinamide Morpholine Acetoacetamide 2-Aminobenzenesulfonic acid 2',3'-cCMP 7 7,8-Dihydrobiopterin Q Glucosamine 6-phosphate Q o-Hydroxyhippuric acid M Q Mucic acid Acetoacetic acid Q p-Aminobenzoic acid Q CDP-choline 7,8-Dihydrofolic acid Glucosamine 6-sulfuric acid Hydroxyindole Maleamic acid cis,cis-Muconic acid Acetoacetyl CoA Q 2-Aminobutyric acid Q110 CDP 7,8-Dihydroneopterin Q Glucosaminic acid Q 5-Hydroxyindoleacetic acid Q Maleic acid Muramic acid Acetohydrazide Q 3-Aminobutyric acid Q110 cGMP Dihydroorotic acid Q110 Glucose 1-phosphate 2-Hydroxyisobutyric acid Q110 Malic acid myo-Inositol 1-phosphate11 Q N-Acetyl-β-alanine 1-Aminocyclopentanecarboxylic acid p-Chlorophenylacetic acid Q110 Glucose 6-phosphate Q 3-Hydroxykynurenine Malonylcarnitine Q myo-Inositol 2-phosphate Q110 Acetyl CoA 1-Aminocyclopropane-1-carboxylic acid Cholic acid Q110 Dihydroxyacetone phosphate Glucurone Q 5-Hydroxylysine Q110 Malonyl CoA myo-Inositol 3-phosphate11 3-Acetylacrylic acid Q 2-Aminoethylphosphonic acid Q110 Choline Q 2,5-Dihydroxybenzoic acid Glucuronic acid Q p-Hydroxymandelic acid Mandelic acid Myosmine Q N-Acetylalanine 1-Aminoethylphosphonic acid Cimetidine 2,4-Dihydroxybenzoic acid trans-Glutaconic acid 5-Hydroxymethyluracil Mannosamine Myristoleic acid Q 4-(β-Acetylaminoethyl)imidazole Q 6-Aminohexanoic acid cIMP 5,6-Dihydroxyindole Q Glutaric acid 6-Hydroxynicotinic acid Q Melatonin N Q N-Acetylasparagine Q 4-Aminohippuric acid trans-Cinnamic acid 3,4-Dihydroxyphenylglycol Glutaryl CoA 2-Hydroxyoctanoic acid 6-Mercaptopurine NAADP Q N-Acetylaspartic acid 5-Aminoimidazole-4-carboxamide ribotide Q Citraconic acid 2,4-Dihydroxypyrimidine-5-carboxylic acid Q110 Glutathione (GSH) 8-Hydroxyoctanoic acid Mesaconic acid Q110 NAD+ Q O-Acetylcarnitine Q 3-Aminoisobutyric acid Q Citramalic acid N,N-Dimethylaniline1 Q110 Glutathione (GSSG) Q 3-(4-Hydroxyphenyl)propionic acid Q110 Met NADH Acetylcholine Q 2-Aminoisobutyric acid Q110 Citric acid 5,6-Dimethylbenzimidazole Q N1-Glutathionylspermidine disulfide Q 3-(2-Hydroxyphenyl)propionic acid Metanilic acid Q110 NADP+ N2-Acetylaminoadipic acid 6-Aminopenicillanic acid Q110 Citrulline Q110 N,N-Dimethylglycine Q Gly-Asp 4-Hydroxyphenylacetaldehyde Methanesulfonic acid NADPH N-Acetylcysteine Q 11-Aminoundecanoic acid CMP-N-acetylneuraminate Q N,N-Dimethylhistidine Q Gly-Gly Q p-Hydroxyphenylacetic acid Q Methionine sulfoxide Neamine N-Acetylgalactosamine o-Aminophenol Q110 CMP 2,5-Dimethylpyrazine Q Gly-Leu o-Hydroxyphenylacetic acid Methionine sulfoximine Q Nicotinamide Q N-Acetylglucosamine p-Aminophenol 3'-CMP9 dIMP Q110 Gly 4-Hydroxyphenylglycine 3-Methoxy-4-hydroxyphenylethyleneglycol Nicotinamide hypoxanthine dinucleotide Q N-Acetylglucosamine 1-phosphate m-Aminophenol 2'-CMP9 Diphenylamine Q110 Glyceraldehyde 3-phosphate 4-Hydroxyphenylglyoxylic acid 5-Methoxy-N,N-dimethyltryptamine Q Nicotine Q N-Acetylglucosamine 6-phosphate 4-Aminophenylsulfone Q110 CoA Q 2,3-Diphosphoglyceric acid Q Glyceric acid p-Hydroxyphenylpyruvic acid 3-Methoxyanthranilic acid Q Nicotinic acid N-Acetylglucosylamine Aminophylline Q p-Coumaric acid dITP Glycerol Q110 Hydroxyproline 5-Methoxyindoleacetic acid p-Nitrophenyl phosphate Q N-Acetylglutamic acid Q 3-Aminopropane-1,2-diol Q o-Coumaric acid Dodecanedioic acid Glycerol 2-phosphate Q cis-4-Hydroxyproline p-Methoxyphenylacetic acid N-Nitrosodiethanolamine Q N-Acetylglutamine 3-Aminopropionitrile Q110 Creatine Q DOPA Q110 Glycerol 3-phosphate Q 3-Hydroxypropionic acid 5-Methoxytryptamine S-Nitrosoglutathione Q N-Acetylglycine Aminopyrine 2-(Creatinine-3-yl)propionic acid Q Dopamine Q Glycerophosphocholine 4-Hydroxyquinoline Q 3-Methoxytyramine 3-Nitrotyrosine Q N-Acetylhistidine 4-Aminosalicylic acid Q110 Creatinine Q dTDP-glucose Glycine anhydride Q 5-Hydroxytryptophan Q 3-Methoxytyrosine Q NMN O-Acetylhomoserine Q 5-Aminovaleric acid Crotonic acid Q110 dTDP Glycocholic acid Hydroxyurea 4-Methyl-2-oxovaleric acid10 Q Noradrenaline Q N-Acetylleucine Q110 AMP cTMP Q110 dTMP Q110 Glycolic acid 2-Hydroxyvaleric acid 3-Methyl-2-oxovaleric acid10 Q Normetanephrine Q N6-Acetyllysine Q 3'-AMP Q110 CTP Q110 dTTP Q N-Glycolylneuraminic acid Q Hypotaurine Q 1-Methyl-4-imidazoleacetic acid Nornicotine N-Acetyllysine Anabasine Cumic acid dUDP Glycyrrhetinic acid Q110 Hypoxanthine Q N1-Methyl-4-pyridone-5-carboxamide Norspermidine N-Acetylmannosamine Aniline β-Cyanoalanine dUMP Q110 Glyoxylic acid I 4-Methyl-5-thiazoleethanol Norvaline Q N-Acetylmethionine Q p-Anisic acid 3',5'-Cyclic dAMP dUTP Q110 GMP Q IDP 4-Methyl-1,2,5-oxadiazol-3-amine O N-Acetylmuramic acid Q Anserine Cyclohexanecarboxylic acid E Q110 GTP Q110 Ile N6-Methyl-2'-deoxyadenosine Octanoic acid Q N-Acetylneuraminic acid Q110 Anthranilic acid Cyclohexylamine Q Ectoine 2-Guanidinobenzimidazole Q Imidazole-4-acetic acid Q 5-Methyl-2'-deoxycytidine Octanoyl CoA

Q N-Acetylornithine Q Arg-Glu Q Cys-Gly Ergothioneine Q 4-Guanidinobutyric acid Imidazole-4-methanol 15-Methyl prostaglandin F2α Q Octanoylcarnitine N-Acetylphenylalanine Q110 Arg Q110 Cys Q110 Erythrose 4-phosphate 3-Guanidinopropionic acid 1H-Imidazole-4-propionic acid Methyl sulfate Octopamine Q N-Acetylputrescine Q Argininosuccinic acid Q Cystathionine Q Ethanolamine Q Guanidinosuccinic acid Q Imidazolelactic acid Q 3-Methyladenine Octopine Q O-Acetylserine Ascorbate 2-glucoside Cysteamine Q Ethanolamine phosphate Q Guanidoacetic acid Q110 IMP N6-Methyladenine Octylamine N-Acetylserine Q Ascorbate 2-sulfate Q Cysteic acid Ethionine Q110 Guanine Indole-3-acetaldehyde Q 1-Methyladenosine Ophthalmic acid Q N1-Acetylspermidine Q Ascorbate 2-phosphate Q Cysteine glutathione disulfide m-Ethoxybenzoic acid Q110 Guanosine Indole-3-acetaldoxim3 Q N-Methylalanine Q110 Ornithine Q N8-Acetylspermidine Ascorbic acid Cysteinesulfinic acid Ethyl glucuronide Guanosine 5'-[γ-thio]triphosphate Q Indole-3-acetamide3 N-Methylaniline Q Q N1-Acetylspermine Q110 Asn Q Cystine Ethylacetimidate Gulonic acid Q Indole-3-acetic acid Q N-Methylanthranilic acid Orotidine 5'-monophosphate Q N-Acetyltryptophan Q110 Asp Q110 Cytidine Q N5-Ethylglutamine Gulonolactone Q Indole-3-ethanol Q Nω-Methylarginine Q Oxamic acid Q110 cis-Aconitic acid Q110 ATP Q110 Cytosine Q N-Ethylglycine H 3-Indolebutyric acid N-Methylaspartic acid 5-Oxo-2-tetrahydrofurancarboxylic acid Q110 Adenine Atrolactic acid D N-Ethylmaleimide Harmaline Q 3-Indoxylsulfuric acid Q threo-β-Methylaspartic acid Q 2-Oxoadipic acid Q110 Adenosine Q Azelaic acid dADP Etidronic acid Q Heptanoic acid Q110 Inosine 2-Methylcholine 2-Oxobutyric acid Adenosine 5'-[γ-thio]triphosphate Azetidine 2-carboxylic acid DAMGO F Hercynine Isatin 3-Methylcrotonyl CoA Q110 2-Oxoglutaric acid Adenosine 5'-phosphosulfate B Daminozide FAD Hexamine Q Isethionic acid Q S-Methylcysteine Q 5-Oxohexanoic acid Q S-Adenosylhomocysteine Barbituric acid dAMP Q Ferulic acid Hexanoic acid Q Isoamylamine Q 5-Methylcytosine 4-Oxohexanoic acid Q110 S-Adenosylmethionine Bentazone Q110 dATP Q FMN Hexylamine Q Isobutylamine Q N-Methylglutamic acid Q110 2-Oxoisovaleric acid Q Adenylosuccinic acid Benzamide dCDP Folic acid S-Hexylglutathione Isobutyric acid S-Methylglutathione 2-Oxooctanoic acid Adipic acid Benzocaine dCMP Formiminoglutamic acid Q Hippuric acid Isobutyryl CoA Q Methylguanidine Q 5-Oxoproline Q ADMA Q Benzoic acid Q110 dCTP Q Formylanthranilic acid Q His-Glu Q Isobutyrylcarnitine 3-Methylguanine Q 4-Oxovaleric acid ADP-glucose5 Q Benzoylformic acid Deamido-NAD+ N-Formylaspartic acid Q110 His Q110 Isocitric acid Q 7-Methylguanine 2-Oxovaleric acid Q ADP-ribose N6-Benzyladenine Q Decanoic acid N-Formylglycine Q Histamine Isoferulic acid Q 1-Methylhistamine P Q110 ADP Benzylsuccinic acid 3-Dehydroshikimic acid Q N-Formylmethionine Q Histidinol Q Isoglutamic acid 3-Methylhistamine Palmatine Q 3',5'-ADP Q110 Betaine Q 5'-Deoxy-5'-methylthioadenosine Q110 Fructose 1,6-diphosphate HMG CoA Q Isonicotinamide 3-Methylhistidine Palmitoylcarnitine Q Adrenaline Q110 Betaine aldehyde 2'-Deoxyadenosine2 Q110 Fructose 6-phosphate Q Homoarginine Q Isonicotinic acid 1-Methylhistidine Q Pantothenic acid Q Agmatine Q Biopterin 5'-Deoxyadenosine2 Q110 Fumaric acid Homocarnosine Isopropanolamine 1-Methylhydantoin Q Paraxanthine Q Ala-Ala Q Biotin Q 2'-Deoxycytidine Fumaric acid monomethyl ester Q Homocitrulline Q 2-Isopropylmalic acid Q N6-Methyllysine Q Pelargonic acid Q β-Ala-Lys Bis(p-nitrophenyl)phosphate 2-Deoxyglucose 6-phosphate 2-Furoic acid Q Homocysteic acid Isovaleric acid Methylmalonic acid Penicillamine Q110 Ala 4-Bromophenylalanine 2'-Deoxyguanosine G Q Homocysteine Isovalerylalanine Methylmalonyl CoA6 Pentamidine Q110 β-Ala Buformin 2-Deoxyribose 1-phosphate Q110 GABA Homocysteinesulfinic acid Q Isovalerylcarnitine S-Methylmethionine Q Perillic acid Albendazole Butyric acid 2'-Deoxyuridine Galactosamine Homocysteinethiolactone Itaconic acid Q 1-Methylnicotinamide Q110 Phe Q Allantoic acid Q γ-Butyrobetaine Q 3'-Dephospho CoA Q Galacturonate 1-phosphate Homocystine ITP Q N-Methylnicotinamide Q Phenaceturic acid Q Allantoin Q Butyrylcarnitine Desthiobiotin Galacturonic acid Q110 Homoserine K N-Methylnorsalsolinol o-Phenanthroline Alliin C dGDP GDP-fucose5 Homoserinelactone Kinetin N-Methylphenylethanolamine Q Phenoxyacetic acid Allocryptopine Q Cadaverine dGMP GDP-galactose8 Q Homovanillic acid Kojic acid Q N-Methylproline Phenoxybenzamine Alloisoleucine Q Caffeine Q dGTP GDP-glucose Hordenine Q Kynurenic acid Q N-Methylputrescine Phenyl phosphate N-Amidinoglutamic acid Q110 cAMP Q N1, N8-Diacetylspermidine GDP-mannose8 Q 3-Hydroxy-3-methylglutaric acid Q Kynurenine 4-Methylpyrazole Q N2-Phenylacetylglutamine Amiloride Canavanine P1, P 4-Di(adenosine-5') tetraphosphate Q110 GDP Q 2-Hydroxy-4-methylvaleric acid L Q 2-Methylserine 1,3-Phenylenediamine 2-Amino-2-(hydroxymethyl)-1,3-propanediol Q N-Carbamoylaspartic acid Q 2,4-Diaminobutyric acid 3',5'-GDP 8-Hydroxy-2'-deoxyguanosine Q110 Lactic acid Q N-Methylserotonin Phenylethanolamine Q 2-Amino-2-methyl-1,3-propanediol Carbamoylphosphate 2,6-Diaminopimelic acid Gibberellic acid Q 3-Hydroxyanthranilic acid Q S-Lactoylglutathione 5-Methyltetrahydrofolic acid 1-Phenylethylamine 2-Amino-2-methylbutyric acid N-Carbamylglutamic acid Q 1,3-Diaminopropane Q110 Gln threo-3-Hydroxyaspartic acid Lauric acid 4-Methylthio-2-oxobutyric acid Q 2-Phenylethylamine Q 4-Amino-3-hydroxybutyric acid β-Carboline 2,3-Diaminopropionic acid γ-Glu-2-aminobutyric acid Q p-Hydroxybenzoic acid Lauroyl CoA (Methylthio)acetic acid Phenylhydrazine 2-Amino-3-phosphonopropionic acid 2-Carboxybenzaldehyde Diethanolamine γ-Glu-Cys Q m-Hydroxybenzoic acid Lauroylcarnitine Q N-Methyltryptamine Q 3-Phenyllactic acid Q 5-Amino-4-oxovaleric acid S-Carboxymethylcysteine Diethylaminomalonic acid Q Glu-Glu Q o-Hydroxybenzoic acid Q Leu-Leu-Tyr Q N-Methyltryptophan Q 3-Phenylpropionic acid 3-Amino-2-piperidone Q Carboxymethyllysine 2,6-Diethylaniline γ-Glu-Val-Gly Q110 3-Hydroxybutyric acid Q110 Leu N-Methyltyramine Phenylpyruvic acid 3-Amino-5-hydroxypyrazole Q Carnitine Q N1, N11-Diethylnorspermine Q110 Glu Q110 2-Hydroxybutyric acid β-Leucine Q Mevalolactone Q Phosphocreatine

10 HMT HMT 11 LC-MS Annotation List Q110 Phosphoenolpyruvic acid Q Sucrose 6'-phosphate Q XMP Mesalamine Q110 6-Phosphogluconic acid Sulfanilic acid XTP Metamitron Q110 3-Phosphoglyceric acid Q S-Sulfocysteine Xylulose 5-phosphate Metaraminol Steroid derivatives Glycochenodeoxycholic acid Luteolin 7-glucoside Acylcarnitine(14:0) Q110 2-Phosphoglyceric acid O-Sulfoserine Z Metformin 11β-Hydroxyandrost-4-ene-3,17-dione Glycocholic acid Malvidin Acylcarnitine(14:1) Q Phosphoglycolic acid Sulfotyrosine Q trans-Zeatin Methotrimeprazine 16-Epiestriol Glycodeoxycholic acid Myricetin 3-rhamnoside Acylcarnitine(14:2) Phosphonoacetic acid Syringic acid DRUGS Methoxamine 16α-Hydroxyestrone Glycolithocholic acid Naringenin Acylcarnitine(14:3) Q Phosphorylcholine T Acetanilide 4,4'-Methylene bis(o-chloroaniline) 17α-Estradiol Glycoursodeoxycholic acid Naringenin 7-neohesperidoside Acylcarnitine(15:0) Q O-Phosphoserine Q Tartaric acid Acetohydroxamic acid 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine 17α-Hydroxypregnenolone12 Lithocholic acid Peonidin 3-glucoside Acylcarnitine(16:1) Phytic acid meso-Tartaric acid Albizziine Q 1-Methyl-2-pyrrolidone 17α-Hydroxyprogesterone Taurochenodeoxycholic acid Poncirin Acylcarnitine(16:2) Picolinamide Q Taurine Amantadine Methysergide 18-Hydroxycorticosterone Taurocholic acid Quercetin Acylcarnitine(17:0) Q Picolinic acid Taurocholic acid Amikacin Metronidazole 19-Hydroxyandrostenedione Taurodeoxycholic acid Quercetin 3-rutinoside Acylcarnitine(17:1) Q Pimelic acid Q Taurocyamine 3-Amino-1,2,4-triazole Q Muscimol 19-Hydroxytestosterone Taurolithocholic acid Resveratrol Acylcarnitine(18:0) Q Pipecolic acid 5,6,7,8-Tetrahydrobiopterin 2-Aminobenzimidazole Nalidixic acid 20α-Hydroxycholesterol13 Taurolithocholic acid 3-sulfate Saponarin Acylcarnitine(18:1) Q Piperidine Tetrahydropalmatine 4-Aminoindole Neomycin B 20α-Hydroxyprogesterone Tauroursodeoxycholic acid Taxifolin Acylcarnitine(18:2) Porphobilinogen Theobromine 5-Aminoindole Neostigmine 21-Deoxycortisol Ursodeoxycholic acid Theaflavin Acylcarnitine(20:0) ppGpp Theophylline (Aminomethyl)phosphonic acid Oleandomycin 21-Hydroxypregnenolone12 Simple/complex lipids Theaflavin-3-gallate Acylcarnitine(20:1) Pristanic acid Thiabendazole Amrinone Oxypurinol 22-Hydroxycholesterol13 N-Acetylsphingosine Fatty acids, acylcarnitines Acylcarnitine(21:0) Q110 Pro Q Thiamine 8-Anilino-1-naphthalenesulfonic acid Pargyline 2-Hydroxyestradiol 1,2-Dipalmitoyl-glycero-3-phosphoethanolamine Arachidic acid Acylcarnitine(22:0) Procaine Thiamine diphosphate Q Arginine ethyl ester Paromomycin 2-Hydroxyestrone 1,2-Dipalmitoyl-glycero-3-phosphoglycerol Arachidonic acid Acylcarnitine(23:0) Q Propionic acid Q Thiamine phosphate Atropine Penciclovir 3α,21-Dihydroxy-5α-pregnan-20-one 1,2-Distearoyl-glycero-3-phosphocholine Behenic acid Others Q Propionyl CoA Q Thiaproline Baclofen Phenazopyridine 4-Androstene-3,17-dione 1-Hexadecyl-2-acetyl-glycero-3-phosphocholine cis-5,8,11,14,17-Eicosapentaenoic acid 5-Methoxyindoleacetic acid Propylthiouracil 6,8-Thioctic acid Benzamidine Phentolamine 5α-Cholestan-3-one 1-Myristoyl-glycero-3-phosphocholine Decanoic acid 6-Hydroxymelatonin

Prostaglandin E2 2-Thiopheneacetic acid Nα-Benzenol arginine ethylester Phenylephrine 5α-Pregnan-20α-ol-3-one 1-Myristoyl-glycero-3-phosphoethanolamine Erucic acid 6-Ketoprostaglandin E1

Prostaglandin F2α Thiourea Benzimidazole Phosphoramidon 5α-Pregnan-3α-ol-20-one 1-Oleoyl-glycero-3-phosphocholine Heneicosanoic acid Abietic acid Q110 PRPP Q Thr-Asp Benzyl viologen Phthalic acid 5α-Pregnane-3,20-dione 1-Palmitoyl-glycero-3-phosphocholine Heptadecanoic acid Astaxanthin Psychosine Q110 Thr Q Betonicine Piperazine 5β-pregnane-3α,21-diol-11,20-dione 1-Palmitoyl-glycero-3-phosphoethanolamine Linoleic acid Biotin Q Pterin Q Threonic acid Carbachol Pirimicarb 7-Dehydrocholesterol 1-Stearoyl-glycero-3-phosphocholine Linolenic acid Carvone Purine allo-Threonine Carbendazim Pralidoxime Adrenosterone Anandamide Myristic acid Dethiobiotin Purine riboside Q110 Thymidine Castanospermine Prazosin Aldosterone Ethyl arachidonate Nervonic acid Indole-3-carboxaldehyde Q110 Putrescine Q110 Chelidonic acid Propiconazole Androstanediol Linoleyl ethanolamide Oleic acid Kynurenic acid Pyrazinamide Tiglic acid Chloramphenicol Propranolol Androstanolone N-Hexanoylsphingosine Palmitic acid N-Acetylserotonin Pyrazole Tioconazole Chloroacetic acid Pseudopelletierine Androsterone Oleoyl ethanolamide Palmitoleic acid N-Acetyltyrosine ethyl ester Pyridine-2-carboxylic acid butyl ester o-Toluic acid 3-Chloroalanine Quisqualic acid Asiatic acid Palmitoylethanolamide Pentadecanoic acid N-Formylanthranilic acid Pyridine p-Toluic acid p-Chlorobenzoic acid Ranitidine Betulinic acid Phytosphingosine Ricinoleic acid p-Anisic acid Q Pyridoxal m-Toluic acid o-Chlorobenzoic acid Rhein Brassinolide Sphinganine Stearic acid Prostaglandin B2 Pyridoxal 5-phosphate Topiramic acid p-Chlorophenoxyacetic acid Ribavirin Campesterol Sphinganine 1-phosphate Stearidonic acid Prostaglandin D1 Q Pyridoxamine Q Trehalose 6-phosphate Ciprofloxacin Ribostamycin Cepharanthine Sphingomyelin(d18:1/16:0) Tricosanoic acid Prostaglandin D2 Q Pyridoxamine 5'-phosphate TRH Clonidine Scopolamine Cholesterol Sphingomyelin(d18:1/18:0) 15(S)-HETE Prostaglandin E1 Q 4-Pyridoxic acid Q Trigonelline 2-Cyanopyridine Q Sebacic acid Cortexolone Sphingosine cis-11,14-Eicosadienoic acid Riboflavin Q Pyridoxine Q Trimethylamine Cycloserine Simazine Corticosterone Sphingosine 1-phosphate cis-11-Eicosenoic acid Thyroxine Pyrimidine Q Trimethylamine N-oxide Cyprodinil Sisomicin Cortisol Stearoyl ethanolamide cis-13-Eicosenoic acid Triptolide Pyrophosphate 4-Trimethylammoniobutanal Daunorubicin Spectinomycin Cortisone Trilaurin cis-4,7,10,13,16,19-Docosahexaenoic acid Tropic acid Pyrrole-2-carboxylic acid Q N6,N6,N6-Trimethyllysine Deethylatrazine Q Stachydrine Cycloartenol Polyphenols cis-8,11,14-Eicosatrienoic acid α-Tocopherol 2,5-Pyrroledione Trimethylsulfonium Deisopropylatrazine Sulfanilamide Dehydroisoandrosterone 7,8-Dihydroxycoumarin Leukotriene B4 α-Tocopherol acetate 1-Pyrroline 5-carboxylic acid Tropic acid 2-Deoxystreptamine Sulfathiazole Dehydroisoandrosterone 3-sulfate 7-Hydroxycoumarin 19-Methylarachidic acid γ-Tocopherol Q110 Pyruvic acid Tropine 2,4-Dichlorobenzoic acid p-Sulfobenzoic acid Deoxycorticosterone Apigenin 7-glucoside 2-Hydroxytetradecanoic acid New Addition (January, 2019) Q Tropinone 2,4-Dichlorophenoxyacetic acid Synephrine Desmosterol Apigenin 8-glucoside 3-Hydroxytetradecanoic acid Cholesterol sulfate Q Quinic acid Q110 Trp Dichlorprop Tacrine Diosgenin Arctigenin Fatty Acid(12:0) 2-Arachidonoylglycerol Quinoline Tryptamine Diclofenac Terbutaline Ergocalciferol Baicalein Fatty Acid(12:1) Acylcarnitine(10:0) 2-Quinolinecarboxylic acid Tryptophanamide 3,5-Diiodotyrosine Terephthalic acid Ergosterol Baicalin Fatty Acid(13:0) AEA(18:1) Q Quinolinic acid Q Tyr-Arg 2,6-Dimethylaniline1 1,2,3,4-Tetrahydroisoquinoline Estriol Capillarisin Fatty Acid(14:1) AEA(18:3) R Q Tyr-Glu 2,4-Dimethylaniline1 Tetrahydrouridine Estriol 16α-glucuronide Catechin Fatty Acid(14:2) AEA(20:3) Q Riboflavin Q110 Tyr 3,5-Dinitrosalicylic acid Timolol Estrone Chlorogenic acid Fatty Acid(14:3) AEA(20:5) Ribose 1-phosphate Q β-Tyr Diphenylcarbazide Tobramycin Estrone 3-glucuronide Chrysin Fatty Acid(15:0) AEA(22:4) Q110 Ribose 5-phosphate Q110 Tyramine Disulfiram Tolazoline Estrone 3-sulfate Chrysoeriol Fatty Acid(15:1) AEA(22:6) Q Ribulose 1,5-diphosphate U Dyphylline Tolmetin Ethynyl estradiol Curcumin Fatty Acid(16:2) Glucosylceramide(d18:1/24:1) Q110 Ribulose 5-phosphate UDP-galactose4 Eflornithine Triadimefon Etiocholan-3α-ol-17-one Daidzein Fatty Acid(16:3) Lactosylceramide(d18:1/24:1) S UDP-glucose4 p-Fluoroaniline 2,4,5-Trichlorophenoxyacetic acid Etiocholan-3α-ol-17-one glucuronide Datiscetin Fatty Acid(17:0) LPA(14:0) Q Saccharopine Q UDP-glucuronic acid o-Fluorobenzoic acid Trientine Hecogenin Delphinidin Fatty Acid(17:1) LPC(12:0) Q110 Sarcosine UDP-N-acetylgalactosamine p-Fluorobenzoic acid Triethanolamine Hydroxyprogesterone caproate Eleutheroside B Fatty Acid(17:2) LPC(24:0) Q SDMA UDP-N-acetylglucosamine m-Fluorobenzoic acid Trifluoperazine Lanosterol Eleutheroside E Fatty Acid(17:3) LPE(16:0) Q110 Sedoheptulose 7-phosphate Q110 UDP Ganciclovir 3,3',5-Triiodothyronine Lathosterol Epicatechin Fatty Acid(19:0) LPE(18:1) Semicarbazide Q110 UMP Gramine Trimethoprim Medroxyprogesterone Epicatechin gallate Fatty Acid(19:1) LPG(16:0) Sepiapterin Undecanoic acid Harman Q Tyrosine methyl ester Methoxyestradiol Epigallocatechin gallate Fatty Acid(19:2) LPG(18:1) Q Ser-Glu Q110 Hexazinone Verapamil Ouabain Eriocitrin Fatty Acid(20:3) LPS(18:0) Q110 Ser Q Urea Hydroxyatrazine Xanthopterin Pregnenolone Eriodictyol Fatty Acid(22:2) LysoPAF(16:0) Q Serotonin Ureidoglycolic acid 2-Hydroxypyridine Pregnenolone sulfate Eriodictyol 7-neohesperidoside Fatty Acid(22:3) PAF(17:0) Shikimate 3-phosphate Q 3-Ureidopropionic acid Ibotenic acid Progesterone Flavanone Fatty Acid(22:4) PGI2 Q Shikimic acid Q Uric acid Ibuprofen Sitosterol Formononetin Fatty Acid(22:5) Retinol Sinapic acid Q110 Uridine 3-Indoleacetonitrile Compounds marked with the same number Solanidine Formononetin 7-glucoside Fatty Acid(24:0) Sphinganine(d20:0) Q Sorbitol 6-phosphate Q Urocanic acid 3-Iodotyrosine at their u pper right a re structural isomers Stigmasterol Galangin Fatty Acid(24:2) Sphingosine(d20:1) Q110 Spermidine Q110 UTP Ismelin which cannot be distinguished as isolated Testosterone Gallocatechin Fatty Acid(24:4) Q110 Spermine V Isoniazid peaks. T he total value of the isomers a re Testosterone acetate Genistein Fatty Acid(24:5) Streptomycin sulfate Q110 Val Isoproterenol reported instead of individual ones. Testosterone glucuronide Glycitein Fatty Acid(25:0) Q Suberic acid Q Valeric acid Isoquinoline β-Estradiol Hesperidin Fatty Acid(25:3) Q110 Succinic acid Q Vanillic acid Kanamycin A β-Estradiol 17-glucuronide Isofraxidin Fatty Acid(26:0) Succinic semialdehyde Q Vanillylmandelic acid Ketoprofen Bile acids Isoliquiritigenin Fatty Acid(26:2) Succinyl CoA6 X Leupeptin Chenodeoxycholic acid Isoorientin Palmitoylcarnitine Succinylcholine Q Xanthine Lidocaine Cholic acid Kaempferol Acylcarnitine(12:0) * Contents in this brochure may be changed without notice. N2-Succinylornithine Q Xanthosine Loperamide Deoxycholic acid Liquiritigenin Acylcarnitine(12:1) Please refer to our website for the latest version of this information. Q O-Succinylhomoserine Q Xanthurenic acid Melamine Hyodeoxycholic acid Luteolin Acylcarnitine(13:1) http://humanmetabolome.com/services/archives

12 HMT HMT 13 Case Study 〈Results（2）〉In Treatment, the ratio of pyluvate to lactate is lower, and amounts of lactate drastically decreased. Lactate (Example): Analysis of cultured cells using C- Scope dehydrogenase (LDH), the enzyme which catalyzes conversion of pyruvate to lactate is known to be under transcriptional 2 Groups; n=3 Blue: DMSO treated group (Control) vs. Red: Anticancer drug-treated group (Treatment) regulation by c-Myc, and the effects of MEK inhibition on the metabolites emerge. 〈Results（3）〉“Total Adenylate” and “Total Guanylate” decreased thanks to the drug treatment. Combined with the 〈Background〉Metabolome analysis provides comprehensive profiling of various small molecules: trends of alterations on an entire metabo- fact AMP, ADP, and ATP decreased as well, that suggests , particularly de novo nucleic acid lism as well as individual rise and fall. In addition to increases and decreases of the metabolites, it can also observe the tendencies with synthesis, was depressed. There are also major drops on: IMP, the precursor of AMP and GMP; PPRP, the starting which the metabolic pathway as a whole is changing. In this case, the effects of an anticancer drug (MARK/ERK kinase [MEK] inhibitor) in material of purine synthesis; and intermediates in the pentose phosphate pathway (PPP), which supplies PPRP. In cultured immortalized mammary gland cells were monitored with C-SCOPE. C-SCOPE specialized for the central energy metabolism the upstream of PPP, 6-phosphogluconate (6-PG) significantly decreased, whereas glucose-6-phosphate (G6P) deeply observes alterations on metabolites essential for survival and reproduction of cells. Here, changes observed in the anticancer didn’t show a big alteration. That indicates the anticancer drug treatment affects activity of factors regulating drug treated group compared to the DMSO treated group (control group) are interpreted through the pathway map in the report. metabolism from G6P to 6-PG, e.g., enzymes such as glucose-6-phosphate dehydrogenase (G6PDH).

Cultured cells n=3; 2 groups Results（1）、（2） Glycolysis DMSO treated group (Control) vs Results（3） Purine Metabolism Anticancer drug treated group (Treatment) The samples were collected 24 hours after the treatment

Pentose Phosphate Pathway Purine Metabolism

Glycolysis

Urea Cycle

Amino Acid Metabolism Polyamine Metabolism TCA cycle

〈Results（1）〉In glycolysis, the difference between Control and Treatment is small on fructose-6-phosphate (F6P), 〈Future works〉The results in this case with C-SCOPE analysis suggest the anticancer drug, an MEK inhibitor, influences the whereas one step after, on fructose-1,6-bisphosphate, a big drop happens in Treatment. The same trend continues in enzymes that regulate glycolysis through suppression of the function of c-Myc located downstream from MEK. In addition, the downstream, indicating activity of phosphofructokinase (PFK), which catalyzes the conversion from F6P to F1,6P, many other unexpected influences on various enzymes and subsequent metabolism were also indicated.Subsequent study is suppressed. It is known that MEK, the target of the anticancer drug used in this case, is located one step before ideas based on the findings in this case are: observation of correlation between abundancy and activity of the targeted c-Myc, a transcription factor regulating transcription of PFK, in the cell signaling pathway. This suggests that enzymes and the drug efficacy, identification of metabolic pathways which may lead to adverse effects then formulating inhibition of MEK leads to inactivation of c-Myc and PFK, and consequently suppresses the entire glycolytic reaction. countermeasures, and evaluation rescue of specific metabolic reactions in the 2nd round metabolome analysis test, etc.

14 HMT HMT 15 HMT’s metabolome analysis service packages Analysis Samples

Sample Types

Biological fluid Plasma, serum, whole blood, blood cells, urine, tears, perspiration, breastmilk, saliva, and cerebrospinal fluid Advanced Scan Basic Scan Dual Scan C-SCOPE Others derived from organisms Liver, kidney, lung, muscle, brain, blood vessel, pancreas, skin, adipose tissue, intestinal mucosa, cultured cells (floating, adherent), stool

Roughly 900 ionic Microorganisms Microbial culture, cultured medium, algae, fungus 116 substances 30 (+24 ) substanc- All metabolites that metabolites + Roughly 900 ionic involved in the es involved in the can be measured roughly 300 Roughly 400 lipid Plants Leaves, stalks, fruits, petals, seeds, algae metabolites central energy central energy by CE-TOFMS lipid-soluble metabolites in total metabolism metabolism metabolites Foods Fermented milk, alcoholic beverages, vegetables, meat, fish, grains, drink products, shellfish

Analysis * We also have a proven track record in the analysis of a variety of other specimens (insects, mollusks, etc.). targets * Depending on the plan, there are also cases where we have not previously analyzed samples even if they are among those listed above. Sugar phosphate, Typical substances Glycometabolism (Addition to Basic Please contact us for details. Ionic metabolites amino acid, nucleic Lipid mediators in metabolism such (glycolysis, acid, organic acid, Scan targets) Fatty tricarboxylic acid (TCA) including (eicosanoids, as sugar vitamins, dipeptides, acids, bile acid, acyl cycle, pentose compounds without lysophospholipids, phosphates, amino short/medium chain carnitine, steroid phosphate pathway), expected formulas etc.), fatty acids, etc. acids, nucleic fatty acids, amino derivatives, etc. adenine nucleotide acid metabolites, etc. acids, etc. (amino acid, urea cycle)

Blood, urine: Blood, urine: Blood: 180 to 200 Blood, culture Estimated 100 to 150 Cultured cells: 200 to 250 Tissue: 250 to 300 medium: 50 to 80 number of Tissue: 200 to 250 Blood: 300 to 350 (Anion) 25-30 Animal and plant Cultured cells: Tissue: 90+ detections* Cultured cells: (Cation) 20-24 tissue: 200 to 400 200 to 300 Cultured cells: 90+ 150 to 200

•Relative area value •Relative area value •Relative area value •Absolute quantita- tive value •Absolute •Absolute •Absolute concentrations of concentrations of concentrations of •Absolute Value of each Reported concentrations in 110 or 403 110 or 403 110 or 403 •Relative area value isotopomer and the values samples (three-point substances in substances in substances in calibration) sum by metabolite samples (one-point samples (one-point samples (one-point •30 metabolic Recommended Sample Amount calibration)** calibration)** calibration)** parameters

•Understanding the •Understanding the •Understanding the impact Volume of sample •Biomarker impact of medicine impact of medicine •Mechanism of medicine discovery using elucidation Type of sample administration and administration and administration and For CE-MS analysis For LC-MS analysis samples of body environment changes, environment changes, focusing on lipid environment changes, fluids such as genetic engineering, and genetic engineering, and metabolism genetic engineering, and Mechanistic Main Blood blood other such factors on other such factors on •Comprehensive other such factors on the studies in basic applications 120 μL 150 μL •Quality assess- the entire primary the entire primary metabolic profiling entire primary metabolism research (plasma, serum) metabolism metabolism ment indices for combined with •Quantitative metabolic •Profiling for components •Profiling for components analysis and long-term food and industrial analysis of ionic Urine 100 μL 100 μL in different food and in different food and metabolites research requiring products, etc. industrial products industrial products absolute quantification

Feces 30 mg 30 mg Metabolite extraction must be performed by customers.* * Results differ depending on the type of analysis sample and on the conditions. ** There will be a charge for additional analyses. The above information is based on specifications as of October 2018. Please refer to “Metabolome Analysis Service Specifications” for details about each plan. Animal tissue 20-40 mg 30-50 mg For adipose tissue, please inquire separately.

Cultured cells 1.0-5.0×106 Cells 1.0-5.0×106 Cells Metabolite extraction must be performed by customers.* Additional options (HepG2, CHO cells, etc.) Cultured cells 1.0-5.0×107 Cells 1.0-5.0×107 Cells Metabolite extraction must be performed by customers.* (blood cells)

Option Description Cultured medium 350 μL 350 μL

Microorganisms Concentrations of the specified 403 metabolites (marked with “Q” on the annotation list”) are Corresponds to 20 mL at OD600 Corresponds to 20 mL at OD600 Metabolite extraction must be performed by customers.* Q-OPTION Premium 400 (Coliform/1 μm diameter) quantified with one-point calibration. Microorganisms Corresponds to 10 mL at OD600 Corresponds to 10 mL at OD600 Metabolite extraction must be performed by customers.* (Yeast/10 μm diameter) Concentration of the specified 110 metabolites (marked with “Q110” on the annotation list”) are Q-OPTION 110 quantified with one-point calibration. Foods (solids) 100 mg 100 mg

LC-TOFMS analyses are performed for the roughly 300 metabolites on the annotation list, and LC-TOFMS analysis Foods (liquids) 1000 μL 1000 μL detected substances are reported.

* Protocols and necessities for sample preprocessing are provided by HMT after orders are placed. Additional analysis report One more pattern of group comparisons, data analyses, and pathway analysis is provided. Important Notice ・Our policy is to specify terms and conditions in contracts. Two-, three-, and four- residue peptides detected in a theoretical value-based database are reported. ・ Peptide Search We may ask customers to perform some pretreatment before sample shipping. (Included in the basic specifications for advanced scan.) In case pretreatment conditions need to be customized, extra cost may be charged. ・The service specifications may be changed without prior notice.

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