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. Author manuscript; available in PMC: 2022 Jul 22.
Published in final edited form as: Methods Mol Biol. 2021;2225:179–197. doi: 10.1007/978-1-0716-1012-1_10

Metabolomics Analysis of Viral Therapeutics

Haiwei Gu 1, Xiaojian Shi 1, Paniz Jasbi 1, Jeffrey Patterson 1
PMCID: PMC9306275  NIHMSID: NIHMS1822360  PMID: 33108663

Abstract

Virotherapy, enabled by recent advances in the transdisciplinary field of biotechnology, has emerged as a powerful tool for use in anticancer treatment, gene therapy, immunotherapy, etc. Examining the effects of viruses and virus-derived immune-modulating therapeutics is of great fundamental and clinical interest. Here we describe a sample preparation protocol for metabolite extraction from virus-infected tissue, in addition to liquid chromatography-mass spectrometry conditions essential for subsequent analysis. This metabolomics approach delivers highly sensitive and specific metabolite information on various biospecimens. Such an approach may be adopted to monitor biological changes in over 30 relevant metabolic pathways in response to viral infection and also viral therapeutics.

Keywords: LC-MS/MS, Metabolomics, Sample preparation, Viral vector, Virotherapy

1. Introduction

There is great interest in the use of viruses and virus-derived components as therapeutics. Viral vectors also hold enormous potential to realize the goals of gene therapy [1] and, in the past decade, more than 1700 gene therapy trials have been enabled by the use of viral therapeutics [2]. In addition to their use as a gene therapy delivery vehicle, viral vectors may also be used as an alternative to antibiotics [3] and, recently, have proven useful in DNA vaccination [4]. Utilization of adeno-associated viruses (AAV) for the deliverance of engineered DNA to target cells [5] has led to the development of fit-for-purpose AAV vectors [6]. Further popularizing such therapeutic concepts is the advent of bacteriophage lambda vectors, or RNA-binding proteins of phage origin [7]. These phage vectors have become an attractive vaccine platform given their inexpensive production, biological stability, and suitability to a wide range of applications with minimal development needed between uses [8]. Additionally, virus-derived immune-modulating proteins may also be used for therapeutic purposes. For example, rabies-derived glycoproteins have been shown to facilitate drug delivery to the central nervous system (CNS) [9], while virus-derived anti-inflammatory proteins may improve the human immune response to immune-mediated diseases and in various cancers through inhibition of protease, chemokine, cytokine, and apoptotic cascades [10].

Since metabolites are sensitive to subtle differences and changes in pathological status and immune response status, metabolomics, the comprehensive study of small-molecular-weight metabolites and their dynamic changes in biological systems [11–15], provides advanced methods to identify changing metabolite levels, and has resulted in the rapid discovery of disease biomarkers during the past decade (see Fig. 1) [16–21]. Mass spectrometry (MS)-based metabolic profiling has proven to be a promising tool for analyzing metabolic alterations due to various diseases and, therefore, can provide sensitive and valuable diagnostic information [18, 22, 23], pathogenesis identification [24, 25], and potential therapeutic targets for clinical treatments [26] and disease monitoring [27]. Indeed, previous studies have applied metabolomics to various aspects of viral therapeutics such as the elucidation of metabolites involved in virus infection and pathogenesis [28] and accurate differentiation of vaccination status [29], in addition to characterizing the metabolic profiles of patients with hepatitis B virus-related hepatocellular carcinoma [30], viruses of the gastrointestinal microbiome [31], inflammatory cytokines involved in H1N1 influenza [32], as well as fingerprinting of HIV-1 and -2 infection in macrophages [33].

Fig. 1.

Fig. 1

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Summary of cancer virotherapy mechanism and LC-MS/MS metabolomics analysis

Herein, we present a sample preparation protocol and associated liquid chromatography-tandem mass spectrometry (LC-MS/MS) conditions for the detection of aqueous metabolites in tissue (see Fig. 2). The targeted LC-MS/MS method used here was modeled and developed after those used in a growing number of studies [18, 34–39].

Fig. 2.

Fig. 2

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Summary of LC-MS/MS sample preparation method

2. Materials

Prepare all solutions using ultrapure water (see Note 1). Ensure that all reagents used are at least analytical grade (≥99% purity). Prepare and store all reagents at room temperature unless indicated otherwise. Follow all pertinent waste disposal protocols as they apply. Thaw samples as directed. Check pipettes to confirm readiness for use. This targeted LC-MS/MS protocol does not provide absolute concentrations of detected metabolites.

2.1. Preparation of Tissue Samples

  1. 1.5 mL Eppendorf tubes.

  2. Bullet beads.

  3. 2 mL glass vials.

  4. 300 μL glass inserts.

  5. 1 mL needle-less syringes.

  6. Polyvinylidene fluoride (PVDF) syringe filters.

  7. Micropipettes with 50–800 μL extraction capability.

  8. 100 mL Methanol (MeOH): 10× dilution with phosphate-buffered saline (PBS).

  9. 100 mL PBS:acetonitrile ([ACN] 4:6 dilution).

  10. Scientific balance with mg readability.

  11. Bullet blender.

  12. Vortex mixer.

  13. Ultrasonic liquid processor.

  14. Centrifuge with speed, time, and temperature control.

  15. Vacuum concentrator.

2.2. LC-MS/MS Targeted Analysis

  1. Suitable ultra-performance liquid chromatography (UPLC) system.

  2. Triple-quadrupole (QQQ)-MS instrument.

  3. Electrospray ionization (ESI) source.

  4. Hydrophilic interaction chromatography (HILIC) column.

  5. Mobile-phase solvents:
    1. 10 mM ammonium acetate, 10 mM ammonium hydroxide in 95% H2O/5% ACN.
    2. 10 mM ammonium acetate, 10 mM ammonium hydroxide in 95% ACN/5% H2O.

3. Methods

In an effort to identify and overcome potential issues, please see Subheading 4.

3.1. Preparation of Tissue Samples

  1. Weigh out 20 mg ± 1.5 mg of biological sample in a 1.5 mL Eppendorf tube.

  2. Homogenize biological sample in 200 μL MeOH:10× diluted PBS in 1.5 mL Eppendorf tube (see Note 2).

  3. Add ½ spoon of beads and disrupt the sample using bullet blender for 2 min.

  4. Centrifuge for 1–2 min to remove liquid from the cap.

  5. Add 800 μL MeOH:10× diluted PBS (see Note 3).

  6. Vortex for 10 s.

  7. Store sample at −20 °C for 30 min.

  8. Sonicate the mixture in an ice bath for 10 min (see Note 4).

  9. Centrifuge the mixture at 21,694 × g for 15 min at 4 °C (see Note 5).

  10. Remove 800 μL supernatant and transfer to a new Eppendorf tube (see Note 6).

  11. Completely dry the sample(s) using a vacuum concentrator at 37 °C for 6 ± 2 h (see Note 7).

  12. Once sample(s) is/are completely dried, reconstitute by adding 200 μL of PBS:ACN solution.

  13. Vortex the sample for 30 s.

  14. Sonicate each sample for 5 min in a room-temperature water bath.

  15. Centrifuge the samples at 21,694 × g for 10 min at 4 °C (see Note 8).

  16. Extract 150 μL of the supernatant into a syringe and slowly plunge the sample through the filter and into a new 1.5 mL Eppendorf tube (see Note 9).

  17. Extract the remaining supernatant from each sample and inject into a single 2 mL tube (larger tubes may be used if needed) for quality control (QC) analysis.

  18. Transfer 100 μL of the filtered experimental sample into a glass insert inside a glass LC vial. The leftover may be used for backup.

  19. Vortex the QC sample for 10 s and then filter it.

  20. Transfer 1 mL of the QC sample into a glass LC vial (see Note 10).

3.2. LC-MS/MS Targeted Analysis

  1. All experiments should be performed using an UPLC-QQQ-MS platform.

  2. Dictate an appropriate worklist such that each sample is injected twice, 10 μL for analysis using negative ionization mode and 4 μL for analysis using positive ionization mode (see Note 11).

  3. The flow rate is maintained at 0.3 mL/min, auto-sampler temperature is kept at 4 °C, while the column compartment is set to 40 °C.

  4. HILIC gradient parameters: After an initial 1-min isocratic elution of 90% mobile phase solvent B, decrease the percentage of solvent B to 40% at t = 11 min. Maintain the composition of solvent B at 40% for 4 min (t = 15 min), after which the percentage of solvent B should gradually be returned to 90%, to prepare for the next injection (see Note 12). An example of a total ion chromatogram (TIC) can be found in Fig. 3.

  5. MS/MS parameters: The mass spectrometer should be equipped with an electrospray ionization (ESI) source. Targeted data acquisition should be performed in multiple reaction monitoring (MRM) mode.

  6. Table 1 lists LC-MS parameters for the validated detection of 310 metabolites using reference standards. For additional parameter details, see Note 13.

Fig. 3.

Fig. 3

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LC-MS/MS total ion chromatogram: (a) negative and (b) positive modes

Table 1.

LC-MS parameters for the validated identification and detection of 310 metabolites

No. HMDB/CAS Compound name Precursorion Production Collision energy Polarity RT (min)
1 HMDB0031645 Acetamide 60.05 42.80 13 Positive 5.050
2 HMDB0001525 Imidazole 69.05 42.00 21 Positive 1.826
3 HMDB0000119 Glyoxylic acid 72.99 44.90 5 Negative 5.031
4 HMDB0001522 Methylguanidine 74.07 57.00 13 Positive 5.730
5 HMDB0000115 Glycolic acid 75.01 46.90 9 Negative 5.350
6 HMDB0014691 Acetohydroxamic acid 76.04 43.00 9 Positive 6.215
7 HMDB0000123 Glycine 76.04 30.10 13 Positive 6.598
8 HMDB0000925 TMAO 76.08 58.00 21 Positive 6.187
9 HMDB0002039 2-Pyrrolidinone 86.06 44.00 29 Positive 1.620
10 HMDB0000243 Pyruvate 87.00 43.00 5 Negative 5.45
11 HMDB0001873 Isobutyric acid 87.04 87.04 5 Negative 1.948
12 HMDB0000190 Lactate 89.02 43.00 9 Negative 4.1
13 HMDB0001414 Putrescine 89.11 72.00 9 Positive 6.118
14 HMDB0000161 Alanine 90.06 44.00 9 Positive 6.140
15 HMDB0000271 Sarcosine 90.06 44.10 9 Positive 6.184
16 HMDB0001882 Dihydroxyacetone 91.04 91.04 0 Positive 1.734
17 HMDB0000005 2-Ketobutyric acid 101.02 57.00 5 Negative 1.490
18 HMDB0000060 Acetoacetate 101.02 56.90 9 Negative 2.194
19 HMDB0002176 2-Methylbutyric acid 101.06 101.06 5 Negative 1.693
20 HMDB0000718 Isovaleric acid 101.06 101.06 5 Negative 6.064
21 HMDB0000691 Malonic acid 103.00 58.90 9 Negative 4.306
22 HMDB0000008 2-Hydroxybutyric acid 103.04 58.90 9 Negative 5.87
23 HMDB0000357 3-Hydroxybutyric acid 103.04 56.80 9 Negative 4.264
24 HMDB0002322 Cadaverine 103.13 85.90 5 Positive 6.506
25 HMDB0001906 2/3-Aminoisobutyric acid 104.07 57.90 9 Positive 5.295
26 HMDB0000452 2-Aminobutyric acid 104.07 57.90 13 Positive 5.706
27 HMDB0031654 3-Aminobutyric acid 104.07 85.90 5 Positive 5.934
28 HMDB0000112 4-Aminobutyric acid 104.07 87.00 9 Positive 6.596
29 HMDB0000092 Dimethylglycine 104.07 57.90 13 Positive 5.295
30 142-26-7 N-Acetylethanolamine 104.07 61.80 5 Positive 1.915
31 HMDB0000097 Choline 104.11 58.00 33 Positive 7.000
32 HMDB0000139 Glyceric acid 105.02 75.00 9 Negative 4.843
33 HMDB0000187 Serine 106.05 60.00 9 Positive 6.824
34 HMDB0001169 4-Aminophenol 110.06 69.00 9 Positive 5.184
35 HMDB0000617 2-Furoic acid 111.01 66.90 5 Negative 1.760
36 HMDB0000630 Cytosine 112.05 94.90 21 Positive 3.445
37 HMDB0000870 Histamine 112.09 95.00 17 Positive 8.673
38 HMDB0000300 Uracil 113.04 71.60 25 Positive 8.707
39 HMDB0000562 Creatinine 114.07 44.10 21 Positive 2.988
40 HMDB0000134 Fumarate 115.00 71.00 5 Negative 2.130
41 HMDB0000176 Maleic acid 115.00 70.90 9 Negative 1.455
42 HMDB0000019 Alpha-ketoisovaleric acid 115.04 70.90 5 Negative 1.455
43 HMDB0000720 Levulinic acid 115.04 70.90 9 Negative 3.250
44 HMDB0000689 4-Methylvaleric acid 115.07 115.07 5 Negative 1.505
45 HMDB0000535 Hexanoic acid 115.07 115.07 5 Negative 1.471
46 HMDB0000162 Proline 116.07 70.00 13 Positive 5.408
47 HMDB0000202 Methylmalonic acid 117.02 73.00 5 Negative 4.622
48 HMDB0000254 Succinate 117.02 73.00 9 Negative 4.293
49 HMDB0000754 Beta-hydroxyisovaleric acid 117.05 71.00 9 Negative 2.132
50 HMDB0000532 Acetylglycine 118.05 75.90 5 Positive 4.335
51 HMDB0000128 Glycocyamine 118.06 42.90 45 Positive 6.344
52 HMDB0000738 Indole 118.07 85.80 13 Positive 5.385
53 HMDB0003355 Amino valerate 118.09 100.90 9 Positive 6.822
54 HMDB0000043 Betaine 118.09 58.00 29 Positive 4.791
55 HMDB0013716 Norvaline 118.09 72.00 5 Positive 5.019
56 HMDB0000883 Valine 118.09 72.00 5 Positive 5.224
57 HMDB0002649 Erythrose 119.03 42.90 17 Negative 4.519
58 HMDB0000719 Homoserine 120.07 73.90 9 Positive 6.343
59 HMDB0000167 Threonine 120.07 73.90 9 Positive 6.343
60 HMDB0011718 4-Hydroxy benzaldehyde 121.03 92.00 25 Negative 1.301
61 HMDB0001870 Benzoic acid 121.03 77.00 9 Negative 1.487
62 HMDB0000574 Cysteine 122.03 80.90 13 Positive 13.454
63 HMDB0001406 Nicotinamide 123.06 79.80 25 Positive 1.746
64 HMDB0001488 Nicotinic acid 124.04 78.10 22 Positive 3.671
65 HMDB0002243 Picolinic acid 124.04 77.70 25 Positive 5.315
66 HMDB0000251 Taurine 126.02 44.10 17 Positive 5.223
67 HMDB0000898 Methylhistamine 126.11 109.10 13 Positive 4.879
68 HMDB0002024 4-Imidazoleacetic acid 127.05 80.90 13 Positive 5.497
69 HMDB0000634 Citraconic acid 129.02 85.10 5 Negative 1.487
70 HMDB0000620 Glutaconic acid 129.02 85.00 5 Negative 4.774
71 HMDB0000491 3-Methyl-2-oxovaleric acid 129.05 129.05 5 Negative 1.351
72 HMDB0000491 Ketoisoleucine 129.05 85.10 5 Negative 1.368
73 HMDB0000695 Ketoleucine 129.05 85.00 5 Negative 1.368
74 HMDB0000267 Pyroglutamic acid 130.05 83.80 9 Positive 4.675
75 HMDB0000070 Pipecolinic acid 130.09 84.00 13 Positive 5.405
76 HMDB0000223 Oxaloacetic acid 131.00 42.00 17 Negative 6.772
77 HMDB0000622 Ethylmalonic acid 131.03 87.00 5 Negative 3.282
78 HMDB0000661 Glutaric acid 131.03 86.90 9 Negative 6.179
79 HMDB0001844 Methyl succinate 131.03 98.90 5 Negative 2.011
80 HMDB0000665 Leucic acid 131.07 85.10 9 Negative 1.994
81 HMDB0001432 Agmatine 131.13 71.90 17 Positive 6.912
82 HMDB0001149 5-Aminolevulinic acid 132.07 85.90 13 Positive 6.521
83 HMDB0000725 Hydroxyproline 132.07 85.90 17 Positive 6.113
84 HMDB0000064 Creatine 132.08 44.00 21 Positive 6.204
85 HMDB0000172 Isoleucine 132.10 86.00 9 Positive 4.697
86 HMDB0000557 l-Alloisoleucine 132.10 86.00 9 Positive 4.468
87 HMDB0000687 Leucine 132.10 86.00 9 Positive 4.468
88 HMDB0001645 Norleucine 132.10 86.00 9 Positive 4.468
89 HMDB0000156 Malate 133.01 114.90 9 Negative 5.87
90 HMDB0000168 Asparagine 133.06 73.90 13 Positive 6.813
91 HMDB0003374 Ornithine 133.10 70.00 21 Positive 11.112
92 HMDB0000191 Aspartate 134.05 73.90 13 Positive 6.471
93 HMDB0000209 Phenylacetic acid 135.04 91.00 5 Negative 1.503
94 HMDB0000034 Adenine 136.06 119.10 25 Positive 2.755
95 HMDB0001895 2-Hydroxybenzoic acid 137.02 93.10 21 Negative 1.215
96 HMDB0000710 4-Hydroxybenzoic acid 137.02 93.10 17 Negative 2.316
97 HMDB0014581 Allopurinol 137.05 54.20 29 Positive 2.070
98 HMDB0000157 Hypoxanthine 137.05 54.80 33 Positive 2.938
99 HMDB0001123 Anthranilic acid 138.06 120.00 9 Positive 1.568
100 20989-17-7 2-Phenylglycinol 138.09 103.00 21 Positive 2.549
101 HMDB0000301 Urocanic acid 139.05 92.90 21 Positive 4.444
102 HMDB0002658 6-Hydroxynicotinic acid 140.04 121.90 17 Positive 4.604
103 HMDB0002349 Muconic acid 141.02 97.00 5 Negative 5.684
104 HMDB0000393 3-Hexenedioic acid 143.03 99.00 5 Negative 6.042
105 HMDB0000482 Caprylic acid 143.11 143.11 5 Negative 1.350
106 HMDB0000208 Alpha-ketoglutaric acid 145.01 101.00 5 Negative 4.99
107 HMDB0000422 2-Methylglutaric acid 145.05 101.10 9 Negative 5.500
108 HMDB0000448 Adipic acid 145.05 101.00 9 Negative 6.025
109 HMDB0000895 Acetylcholine 146.12 86.80 9 Positive 3.439
110 HMDB0001257 Spermidine 146.17 72.00 17 Positive 6.499
111 HMDB0059655 2HG 147.03 129.00 5 Negative 5.49
112 HMDB0000641 Glutamine 147.08 84.00 17 Positive 6.673
113 HMDB0000182 Lysine 147.12 83.90 21 Positive 11.749
114 HMDB0003339 Glutamic acid 148.06 84.00 17 Positive 6.39
115 147-73-9 Meso-tartaric acid 149.01 86.90 13 Negative 11.000
116 HMDB0059916 Tartaric acid 149.01 87.00 9 Negative 11.000
117 HMDB0001587 Phenylglyoxylic acid 149.02 77.00 9 Negative 1.315
118 HMDB0000646 l-(+)-Arabinose 149.04 59.00 20 Negative 6.034
119 HMDB0000283 Ribose 149.04 89.00 5 Negative 4.4
120 HMDB0000098 Xylose 149.04 89.00 5 Negative 4.320
121 HMDB0002097 4-Ethylbenzoic acid 149.06 105.10 9 Negative 1.501
122 HMDB0032075 p-Tolylacetic acid 149.06 105.00 5 Negative 1.501
123 HMDB0001878 Thymol 149.09 87.00 13 Negative 11.100
124 HMDB0000696 Methionine 150.06 103.90 9 Positive 4.923
125 HMDB0000669 2-Hydroxy phenylacetic acid 151.04 107.00 13 Negative 1.280
126 HMDB0000440 3-Hydroxyphenylacetic acid 151.04 107.00 5 Negative 1.280
127 HMDB0004815 4-Hydroxy-3-methylbenzoic acid 151.04 107.10 9 Negative 1.923
128 HMDB0000020 4-Hydroxyphenylacetic acid 151.04 107.00 9 Negative 1.907
129 HMDB0000703 Mandelic acid 151.04 107.00 5 Negative 1.907
130 HMDB0000508 Adonitol 151.06 70.80 16 Negative 4.149
131 HMDB0001851 L-(—)-Arabitol 151.06 89.10 8 Negative 4.335
132 HMDB0002917 Xylitol 151.06 59.00 20 Negative 4.390
133 HMDB0000132 Guanine 152.06 135.10 21 Positive 4.009
134 HMDB0000397 2,3-Dihydroxybenzoic acid 153.02 109.00 17 Negative 1.027
135 HMDB0000152 Gentisic acid 153.02 108.10 25 Negative 1.415
136 HMDB0001856 Protocatechuic acid 153.02 109.00 13 Negative 1.731
137 HMDB0000292 Xanthine 153.04 109.90 21 Positive 3.415
138 56-17-7 Cystamine 153.05 107.70 5 Positive 9.759
139 HMDB0001476 3-Hydroxyanthranilic acid 154.05 112.90 5 Positive 13.765
140 HMDB0000073 Dopamine 154.09 137.00 9 Positive 4.015
141 HMDB0000177 Histidine 156.08 110.10 13 Positive 7.182
142 HMDB0000555 3-Methyladipic acid 159.06 97.20 9 Negative 5.464
143 HMDB0000678 Isovalerylglycine 160.10 57.00 17 Positive 2.889
144 HMDB0000303 Tryptamine 161.11 144.00 9 Positive 3.433
145 HMDB0000510 2-Aminoadipic acid 162.08 98.00 13 Positive 6.839
146 HMDB0000062 Carnitine 162.12 43.00 25 Positive 6.550
147 HMDB0001713 m-Coumaric acid 163.04 119.00 17 Negative 1.499
148 HMDB0002035 p-Coumaric acid 163.04 119.00 17 Negative 1.849
149 HMDB0000205 Phenylpyruvic acid 163.04 91.00 9 Negative 1.279
150 HMDB0001890 Acetylcysteine 164.04 123.00 9 Positive 2.555
151 HMDB0002107 Phthalic acid 165.02 121.00 5 Negative 2.380
152 HMDB0060256 d-Xylonic acid 165.04 75.00 16 Negative 5.218
153 HMDB0000779 3-Phenyllactic acid 165.05 147.10 9 Negative 1.499
154 HMDB0002072 4-Methoxyphenylacetic acid 165.05 106.00 9 Negative 1.499
155 HMDB0000159 Phenylalanine 166.09 120.20 17 Positive 4.373
156 HMDB0000263 Phosphoenolpyruvic acid 166.97 78.80 33 Negative 6.060
157 HMDB0000130 Homogentisic acid 167.03 108.00 13 Negative 5.765
158 HMDB0000484 Vanillic acid 167.03 152.00 13 Negative 2.000
159 HMDB0000232 Quinolinic acid 168.03 150.00 5 Positive 5.263
160 HMDB0001112 d-Glyceraldehyde 3-phosphate 168.99 79.00 33 Negative 5.800
161 HMDB0000289 Urate 169.04 141.00 13 Positive 4.768
162 HMDB0000239 Pyridoxine 170.08 134.10 17 Positive 2.295
163 HMDB0000001 1-Methylhistidine 170.10 95.90 21 Positive 6.930
164 HMDB0000511 Capric acid 171.14 171.14 5 Negative 1.261
165 HMDB0000072 Aconitic acid 173.01 85.00 9 Negative 6.191
166 HMDB0003070 Shikimic acid 173.04 93.10 9 Negative 5.801
167 HMDB0000893 Suberic acid 173.08 111.10 13 Negative 5.004
168 HMDB0000721 Glycylproline 173.09 116.10 13 Positive 6.701
169 HMDB0000044 l-Ascorbic acid 175.02 114.90 9 Negative 3.027
170 HMDB0003357 Acetylornithine 175.11 70.00 25 Positive 6.769
171 HMDB0000517 Arginine 175.12 70.00 25 Positive 11.665
172 HMDB0000197 Indole-3-acetic acid 176.07 158.10 13 Positive 6.193
173 HMDB0000904 Citrulline 176.11 69.90 25 Positive 7.020
174 HMDB0000259 Serotonin 177.10 160.00 9 Positive 4.326
175 HMDB0000707 4-Hydroxy phenylpyruvic acid 179.03 135.00 9 Negative 1.273
176 HMDB0000660 Fructose 179.05 89.00 5 Negative 4.716
177 HMDB0033704 Galactose 179.05 89.10 5 Negative 4.732
178 HMDB0000122 Glucose 179.05 89.00 5 Negative 5.410
179 HMDB0000169 Mannose 179.05 58.90 17 Negative 5.350
180 HMDB0000211 Myoinositol 179.05 135.00 1 Negative 1.497
181 HMDB0000714 Hippuric acid 180.07 104.90 13 Positive 2.499
182 HMDB0006479 Glucosamine 180.09 162.00 5 Positive 6.791
183 HMDB0000118 Homovanillic acid 181.05 136.80 5 Negative 2.268
184 HMDB0003269 Nicotinuric acid 181.06 134.80 17 Positive 4.234
185 HMDB0000765 D-Mannitol 181.07 101.10 20 Negative 5.213
186 HMDB0000107 Dulcitol 181.07 58.90 24 Negative 5.294
187 HMDB0000247 Sorbitol 181.07 58.70 17 Negative 5.223
188 HMDB0000158 Tyrosine 182.08 90.90 33 Positive 5.284
189 HMDB0000017 4-Pyridoxic acid 184.06 166.10 9 Positive 1.288
190 HMDB0000068 Epinephrine 184.10 166.10 5 Positive 5.366
191 HMDB0000819 Normetanephrine 184.10 166.10 5 Positive 4.462
192 HMDB0000807 3-Phosphoglyceric acid 184.98 79.00 50 Negative 7.013
193 HMDB0000784 Azelaic acid 187.09 125.10 13 Negative 4.020
194 HMDB0006029 Acetyl-l-glutamine 189.09 130.00 13 Positive 4.651
195 HMDB0000715 Kynurenic acid 190.05 144.00 25 Positive 2.338
196 HMDB0002302 3-Indolepropionic acid 190.09 130.10 25 Positive 1.562
197 HMDB0000094 Citrate 191.02 111.00 9 Negative 7
198 HMDB0000193 Isocitrate 191.02 111.00 13 Negative 6.41
199 HMDB0000763 HIAA 192.07 146.10 13 Positive 4.507
200 HMDB0002545 d-Galacturonic acid 193.03 58.70 17 Negative 6.662
201 HMDB0000127 Glucuronic acid 193.03 113.00 9 Negative 6.493
202 HMDB0000954 Ferulic acid 193.05 134.00 13 Negative 1.512
203 HMDB0029965 Methyl alpha-d-glucopyranoside 193.07 102.80 13 Negative 4.320
204 HMDB0029965 Methyl-d-mannopyranoside 193.07 58.80 21 Negative 3.044
205 HMDB0000565 Galactonic acid 195.05 75.00 21 Negative 6.831
206 HMDB0000625 Gluconic acid 195.05 129.00 13 Negative 6.900
207 HMDB0001847 Caffeine 195.09 138.00 17 Positive 1.447
208 HMDB0000609 DOPA 198.08 152.10 5 Positive 5.838
209 HMDB0004063 Metanephrine 198.12 180.10 5 Positive 3.959
210 HMDB0000624 Lauric acid 199.17 199.17 5 Negative 1.224
211 HMDB0000792 Sebacic acid 201.11 139.10 17 Negative 3.612
212 HMDB0003334 Dimethylarginine 203.15 70.00 21 Positive 10.306
213 HMDB0060484 Indole-3-pyruvic acid 204.07 163.20 5 Positive 12.162
214 HMDB0000201 Acetylcarnitine 204.13 85.00 17 Positive 5.661
215 HMDB0000929 Tryptophan 205.10 187.90 5 Positive 4.437
216 HMDB0000881 Xanthurenic acid 206.05 131.90 29 Positive 2.245
217 HMDB0000671 Indole-3-lactic acid 206.08 118.10 25 Positive 4.453
218 HMDB0000639 Mucic acid 209.03 84.90 13 Negative 7.078
219 HMDB0000684 Kynurenine 209.09 192.00 5 Positive 4.483
220 HMDB0001511 Phosphocreatine 212.05 45.00 17 Positive 1.674
221 2280-85-5 6-Methyl-dl-tryptophan 219.12 202.10 5 Positive 4.071
222 HMDB0000210 Pantothenic acid 220.12 90.00 9 Positive 4.071
223 HMDB0000472 5-Hydroxytryptophan 221.09 204.00 5 Positive 5.236
224 HMDB0000215 Acetylglucosamine 222.10 138.10 17 Positive 4.505
225 HMDB0000853 N-Acetyl-d-galactosamine 222.10 204.10 4 Positive 4.387
226 HMDB0000215 N-Acetyl-d-glucosamine 222.10 204.30 0 Positive 4.487
227 HMDB0000742 Homocysteine 223.08 88.00 29 Positive 8.604
228 HMDB0000732 3-Hydroxykynurenine 225.09 207.00 5 Positive 1.102
229 2387-23-7 N,N-Dicyclohexylurea 225.20 100.10 13 Positive 1.239
230 HMDB0001904 3-Nitrotyrosine 227.07 181.00 5 Positive 4.801
231 HMDB0000033 Carnosine 227.12 110.00 21 Positive 7.655
232 HMDB0000806 Myristic acid 227.20 227.20 5 Negative 1.189
233 HMDB0000014 2-Deoxycytidine 228.10 112.00 9 Positive 3.591
234 4300-28-1 d-Ribose 5-phosphate 229.01 78.80 45 Negative 6.850
235 66768-39-6 d-Xylose 5-phosphate 229.01 78.90 45 Negative 5.200
236 HMDB0000012 2-Deoxyuridine 229.08 113.00 9 Positive 2.289
237 HMDB0001923 Naproxen 229.08 169.00 33 Negative 1.358
238 HMDB0014732 Amiloride 230.06 171.00 13 Positive 4.192
239 HMDB0001389 Melatonin 233.13 174.00 13 Positive 1.307
240 HMDB0000192 Cystine 241.03 74.00 21 Positive 7.980
241 HMDB0000826 Pentadecanoic acid 241.21 241.21 5 Negative 1.189
242 HMDB0000089 Cytidine 244.10 112.00 9 Positive 4.229
243 HMDB0000296 Uridine 245.08 113.00 5 Positive 2.950
244 HMDB0000030 Biotin 245.10 227.00 9 Positive 3.955
245 HMDB0000101 2-Deoxyadenosine 252.11 136.10 13 Positive 2.585
246 HMDB0000845 Neopterin 254.09 206.00 21 Positive 5.621
247 HMDB0000220 Palmitic acid 255.23 255.23 5 Negative 1.189
248 HMDB0000982 5-Methylcytidine 258.11 126.10 9 Positive 3.932
249 HMDB0001078 d-Mannose 6-phosphate 259.02 79.10 52 Negative 7.170
250 HMDB0000124 Fructose 6-phosphate 259.02 78.80 41 Negative 6.87
251 HMDB0001586 Glucose 1-phosphate 259.02 79.10 29 Negative 6.889
252 HMDB0001401 Glucose 6-phosphate 259.02 78.80 41 Negative 7.300
253 HMDB0001254 Glucosamine 6-phosphate 260.06 126.10 9 Positive 7.177
254 HMDB0001849 Propranolol 260.17 116.00 17 Positive 2.516
255 94-24-6 Tetracaine 265.19 176.00 17 Positive 1.534
256 HMDB0000085 2-Deoxyguanosine 268.11 151.90 17 Positive 4.000
257 HMDB0000050 Adenosine 268.11 136.00 13 Positive 3.018
258 HMDB0000195 Inosine 269.09 137.00 21 Positive 3.794
259 HMDB0002259 Heptadecanoic acid 269.25 269.25 5 Negative 1.154
260 HMDB0001316 6-Phosphogluconic
acid		 275.01 78.90 49 Negative 13.882
261 506-24-1 9-Octadecynoic acid 279.23 279.23 5 Negative 1.188
262 HMDB0003331 1-Methyladenosine 282.12 150.00 29 Positive 6.397
263 HMDB0000827 Stearic acid 283.26 283.26 5 Negative 1.137
264 HMDB0000133 Guanosine 284.10 152.10 13 Positive 4.616
265 HMDB0000299 Xanthosine 285.09 153.00 9 Positive 4.844
266 HMDB0060493 N-Acetylmuramic acid 292.10 89.00 8 Negative 3.895
267 HMDB0000772 Nonadecanoic acid 297.28 297.28 5 Negative 1.120
268 HMDB0001409 dUMP 307.03 195.10 17 Negative 7.235
269 HMDB0000125 Glutathione reduced 308.09 84.10 25 Positive 6.070
270 HMDB0014950 Phenylbutazone 309.16 76.90 45 Positive 1.030
271 HMDB0000230 N-Acetylneuraminic acid 310.12 274.00 5 Positive 6.350
272 HMDB0000651 Decanoylcarnitine 316.25 84.90 25 Positive 2.332
273 HMDB0001227 dTMP 323.07 80.80 21 Positive 6.033
274 HMDB0001058 Fructose 1,6 biphosphate (F16BP) 338.99 78.90 49 Negative 7.9
275 HMDB0003514 Glucose 1,6 biphosphate (G16BP) 338.99 241.10 17 Negative 8.000
276 HMDB0000055 d-(+)-Cellobiose 341.11 161.00 4 Negative 6.869
277 HMDB0005826 Galactinol dihydrate 341.11 179.20 16 Negative 8.318
278 HMDB0000186 Lactose 341.11 161.00 5 Negative 7.065
279 HMDB0000258 Sucrose 341.11 59.00 50 Negative 6.474
280 HMDB0000975 Trehalose 341.11 178.90 9 Negative 7.014
281 HMDB0003559 Gibberellic acid 345.13 143.00 37 Negative 1.559
282 HMDB0001314 cGMP 346.06 152.00 21 Positive 4.63
283 HMDB0000045 AMP 348.10 136.20 17 Positive 6.544
284 HMDB0001220 Prostaglandin E2 351.21 315.20 9 Negative 1.474
285 HMDB0000939 Adenosyl-l-homocysteine 385.13 136.00 25 Positive 6.875
286 HMDB0001245 DCDP 388.03 112.00 17 Positive 6.922
287 HMDB0000774 Pregnenolone sulfate 395.19 96.80 45 Negative 1.017
288 HMDB0000295 UDP 402.99 78.90 50 Negative 6.79
289 HMDB0001341 ADP 428.04 136.10 21 Positive 6.900
290 HMDB0000121 Folic acid 442.15 295.00 25 Positive 6.806
291 HMDB0001201 GDP 444.03 152.10 33 Positive 7.255
292 HMDB0001056 Dihydrofolic acid 444.17 297.10 17 Positive 6.783
293 HMDB0000797 SAICAR 455.08 110.00 44 Positive 6.947
294 HMDB0000536 Adenylosuccinate 464.08 252.00 25 Positive 8.036
295 HMDB0000998 dCTP 468.00 111.90 17 Positive 7.5
296 HMDB0001191 dUTP 468.98 80.90 13 Positive 7
297 HMDB0001562 Folinic acid 474.18 327.10 17 Positive 1.464
298 HMDB0003213 Raffinose 503.16 178.90 25 Negative 7.912
299 HMDB0000538 ATP 508.01 136.00 45 Positive 7.350
300 HMDB0001178 ADP ribose 560.10 136.20 21 Positive 6.020
301 HMDB0000290 UDP-GlcNAc 608.09 204.30 9 Positive 7.010
302 HMDB0003337 Glutathione oxidized 613.16 484.00 17 Positive 7.267
303 HMDB0000902 NAD 664.12 136.00 41 Positive 6.767
304 HMDB0003553 Stachyose hydrate 665.21 383.20 40 Negative 8.960
305 HMDB0001487 NADH 666.10 348.10 17 Positive 5.700
306 HMDB0000217 NADP 745.10 604.10 17 Positive 7.200
307 HMDB0000221 NADPH 746.10 746.10 17 Positive 6.500
308 HMDB0001206 Acetyl-CoA 810.14 303.10 37 Positive 6
309 HMDB0001243 Isobutyryl-CoA 838.17 331.20 37 Positive 7.000
310 HMDB0001166 Hydroxybutyryl coenzyme A 854.16 347.10 37 Positive 6.954
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4. Notes

  1. Render ultrapure water by filtering deionized water.

  2. 4:6 dilution of PBS:ACN contains 4:1 (v:v) solution of 13C-lactate and 13C-glutamic acid.

  3. Added solutions must be cooled at 4 °C.

  4. Prepare ice for use prior to sonication.

  5. Ensure that the centrifuge is balanced.

  6. Resulting protein pellets may be extracted and saved for protein analysis as needs arise.

  7. Drying time is contingent on the number of samples and type of tissue used. It is permitted to dry samples overnight. If the extracted supernatant cannot be dried immediately, samples may be kept at −20 °C for short-term storage or at −80 °C for long-term storage.

  8. During this time, you may prepare the vial inserts and syringe filters.

  9. It may be necessary to plunge multiple times in order to filter all of the sample. At least 50 μL of filtered sample is required for LC-MS/MS analysis.

  10. A glass insert is not needed for analysis of QC sample given sufficient volume of liquid.

  11. Both chromatographic separations should be performed in HILIC mode.

  12. Targeted data acquisition should be performed in multiple-reaction-monitoring (MRM) mode.

  13. MS parameters were optimized using standard references. All LC separation was performed using basic HILIC. The dwell time should be configured to 5 s for each ion signal. Unit resolution is required for both MS1 and MS2.

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