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Targeted metabolomics

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Targeted metabolomics

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Targeted metabolomics analysis is the targeted and specific detection and analysis of the specified metabolites in the list by using standard samples as controls.

The key points of targeted metabolomics analysis are high accuracy, high throughput and high reliability. Although this type of analysis is more routine than metabolic profiling, it can be used for very large numbers of samples.

Targeted metabolomics can be applied to.

(1) Validating new findings and hypotheses proposed by metabolomics experiments;

(2) Conducting hypothesis-based exploratory experiments and studying metabolic models for specific metabolites.

Basic Research Process

The process of targeted metabolomics research can be divided into: sample collection, standard curve creation, metabolite extraction and isolation, on-line assay, and data analysis.

Sample types

Serum - Plasma - Urine - Cerebrospinal fluid - Saliva - Feces - Microorganisms - Cells - Plants - Tissues - Others

Targeted metabonomics services and cycles are shown in the following table:

**靶向代谢组学服务和周期**
平台	仪器型号	代谢物	化合物数目	检测周期	检测价格
GC-MS/MS	Aglient 7890A-5975C或8890-7010B	短链脂肪酸	11种短链脂肪酸	约15个工作日	请询价
GC-MS/MS	Aglient 7890A-5975C或8890-7010B	游离脂肪酸	37种游离脂肪酸	约15个工作日	请询价
UHPLC-QqQ MS	SCIEX QTRAP 4500或Triple Quad 4500	维生素	12种水溶性	约20个工作日	请询价
		氨基酸	32种氨基酸
		神经递质	27种神经递质
		植物激素	10种植物激素
		胆汁酸	48种胆汁酸
		辅酶	18种辅酶
		抗生素	105种抗生素

**11种短链脂肪酸绝对定性定量分析**
序号	中文名称	英文名称	分子式	CAS号	相对分子质量
1	乙酸	acetic acid	C₂H₄O₂	64-19-7	60.05
2	丙酸	propionic acid	C₃H₆O2	79-09-4	74.08
3	异丁酸	isobutyric acid	C₄H₈O₂	79-31-2	88.11
4	丁酸	n-butyric acid	C₄H₈O₂	107-92-6	88.11
5	异戊酸	isovaleric acid	C₅H₁₀O₂	503-74-2	102.13
6	戊酸	n-valeric acid	C₅H₁₀O₂	109-52-4	102.13
7	4-甲基戊酸	4-methylvaleric acid	C₆H₁₂O₂	646-07-1	116.16
8	正己酸	hexanoic acid	C₆H₁₂O₂	142-62-1	116.15
9	2-甲基己酸	2-methylhexanoic acid	C₇H₁₄O₂	4536-23-6	130.18
10	正庚酸	n-heptylic acid	C₇H₁₄O₂	111-14-8	130.18
11	正辛酸	n-octanoic acid	C₈H₁₆O₂	124-07-2	144.21

**37种游离脂肪酸绝对定性定量分析**
编号	中文名称	英文名称	简写
1	丁酸	butyric acid	C4:0
2	己酸	caproic acid	C6:0
3	辛酸	caprylic acid	C8:0
4	癸酸	capric acid	C10:0
5	十一烷酸	undecanoic acid	C11:0
6	月桂酸	lauric acid	C12:0
7	十三烷酸	tridecanoic acid	C13:0
8	十四酸	myristic acid	C14:0
9	十四碳烯酸	myristoleic acid	C14:1n5
10	十五烷酸	pentadecanoic acid	C15:0
11	顺-10-十五碳烯酸	cis-10-pentadecenoic acid	C15:1n5
12	棕榈酸	palmitic acid	C16:0
13	棕榈油酸	palmitoleic acid	C16:1n7
14	十七烷酸	heptadecanoic acid	C17:0
15	顺-10-十七烯酸	cis-10-heptadecenoic acid	C17:1n7
16	硬脂酸	stearic acid	C18:0
17	反油酸	elaidic acid	C18:1n9t
18	油酸	oleic acid	C18:1n9c
19	反亚油酸	linolelaidic acid	C18:2n6t
20	亚油酸	linoleic acid	C18:2n6c
21	γ-亚麻酸	r-linolenic acid	C18:3n6
22	α-亚麻酸	α-linolenic acid	C18:3n3
23	花生酸	arachidic acid	C20:0
24	顺-11-二十碳烯酸	cis-11-eicosenoic acid	C20:1
25	顺-11,14-二十碳二烯酸	cis-11,14-eicosadienoic acid	C20:2
26	顺-8,11,14-二十碳三烯酸	cis-8,11,14-eicosatrienoic acid	C20:3n6
27	花生四烯酸	arachildonic acid	C20:4n6
28	二十一烷酸	heneicosanoic acid	C21:0
29	顺-11,14,17-二十碳三烯酸	cis-11,14,17-eicosatrienoic acid	C20:3n3
30	顺-5,8,11,14,17-二十碳三烯酸	cis-5,8,11,14,17-eicosatrienoic acid	C20:5n3
31	山萮酸	behenic acid	C22:0
32	芥酸	erucic acid	C22:1n9
33	顺-13,16-二十二碳二烯酸	cis-13,16-docosadienoic acid	C22:2
34	三十烷酸	tricosanoic acid	C23:0
35	顺-4,7,10,13,16,19-二十二碳六烯酸	cis-4,7,10,13,16,19-docosahexaenoic acid	C22:6n3
36	二十四碳酸	lignceric acid	C24:0
37	神经酸	nervonic acid	C24:1n9

**12种水溶性维生素绝对定性定量分析**
编号	物质名称	英文名称	分子量
1	VB1硫胺素	thiamine	264.35
2	VB2核黄素	Riboflavin	376.36
3	VB3烟酰胺	Nicotinamide	122.12
4	VB5泛酸	(R)-pantothenicacid	219.24
5	VB6吡哆胺	pyridoxamine	168.19
6	VB6吡哆醇	Pyridoxine	169.18
7	VB6吡哆醛	pyridoxal	167.16
8	VB7生物素	Biotin	244.31
9	VB9叶酸	folic acid	441.40
10	VB12钴胺素碎片	Vitamin B12	1355.37
11	对氨基苯甲酸	p-aminobenzoic acid	137.14
12	硫辛酸	thioctic acid	206.33

**32种氨基酸绝对定性定量分析**
编号	中文名称	英文名称	缩写
1	丙氨酸	alanine	ALA
2	肌氨酸	sarcosine	SAR
3	甘氨酸	glycine	GLY
4	α-氨基丁酸	α-aminobutyric acid	ABA
5	缬氨酸	valine	VAL
6	β-氨基异丁酸	β-aminobutyric acid	β-AIB
7	亮氨酸	leucine	LEU
8	别异亮氨酸	allo-isoleucine	aILE
9	异亮氨酸	isoleucine	ILE
10	苏氨酸	threonine	THR
11	丝氨酸	serine	SER
12	脯氨酸	proline	PRO
13	天冬酰胺	asparagine	ASN
14	硫代脯氨酸	thioproline	TPR
15	天冬氨酸	aspartic acid	ASP
16	蛋氨酸	methionine	MET
17	羟基脯氨酸	4-hydroxyproline	4-HYP
18	谷氨酸	glutamic acid	GLU
19	苯丙氨酸	phenylalanine	PHE
20	α-氨基己二酸	ɑ-aminoadipic acid	AAA
21	α-氨基庚二酸	ɑ-aminopimelic acid	APA
22	谷氨酰胺	glutamine	GLN
23	鸟氨酸	ornithine	ORN
24	甘氨酰-脯氨酸（二肽）	glycyl-proline	GPR
25	赖氨酸	lysine	LYS
26	组氨酸	histidine	HIS
27	羟基赖氨酸	hydroxylysine	HLY
28	酪氨酸	trrosinre	TYR
29	脯氨酸-羟基脯氨酸（二肽）	proline-hydroxyproline	PHP
30	色氨酸	tryptophan	TRP
31	胱硫醚	cystathionine	CTH
32	胱氨酸	cystine	C-C

**27种神经递质绝对定性定量分析**
编号	中文名字	英文名字	缩写	分类	CAS号
1	色胺	Tryptamine	-	单胺类吲哚类	61-54-1
2	5-羟色胺	Serotonin hydrochloride	5-HT	单胺类吲哚类	153-98-0
3	5-羟吲哚乙酸	5-hydroxyindoleacetic acid	5HIAA	单胺类吲哚类	54-16-0
4	犬尿喹啉酸	Kynurenic acid	KYNA	单胺类吲哚类	492-27-3
5	褪黑激素	Melatonin	-	单胺吲哚类	73-31-4
6	5-羟基色氨酸	5-Hydroxy-Tryptophan	5-HTP	单胺吲哚类	4350-09-8
7	肾上腺素	Epinephrine	EP	单胺类儿茶酚胺类	329-63-5
8	去甲肾上腺素	noradrenaline bitartrate	NE	单胺类儿茶酚胺类	108341-18-0
9	多巴胺	Dopamine	DA	单胺类儿茶酚胺类	62-31-7
10	高香草酸	homovanillic acid	NMN	单胺类儿茶酚胺类	306-08-1
11	香草扁桃酸	4-hydroxy-S-methoxymandelic acid	VM-A	单胺类儿茶酚胺类	55-10-7
12	去甲变肾上腺素	rac Normetanephrine	-	单胺类儿茶酚胺类	1011-74-1
13	3-甲氧酪胺	3-Methoxy-p-tyramine	-	单胺类儿茶酚胺类	1477-68-5
14	酪胺	Tyramine	-	单胺类儿茶酚胺类	51-67-2
15	二羟苯乙酸	3,4-Dihydroxyphenylacetic acid	DOPAC	单胺类儿茶酚胺类	102-32-9
16	胆碱	Choline Chloride	-	单胺类胆碱类	67-48-1
17	乙酰胆碱	Acetylcholine	Ach	单胺类胆碱类	60-31-1
18	组胺	Histamine	-	氨基酸类	51-45-6
19	犬尿氨酸	Kynurenine	-	氨基酸类	2922-83-0
20	谷氨酰胺	Glutamine	GLN	氨基酸类	56-85-9
21	谷氨酸	Glutamic Acid	GLU	氨基酸类	56-86-0
22	4-氨基丁酸	4-Aminobutyric Acid	GABA	氨基酸类	56-12-2
23	酪氨酸	Tyrosine	TYR	氨基酸类	60-18-4
24	精氨酸	Arginine	Arg	氨基酸类	74-79-3
25	组氨酸	Histidine	HIS	氨基酸类	71-00-1
26	苯基丙氨酸	Phenylalanine	PHE	氨基酸类	63-91-2
27	色氨酸	Tryptophan	TRP	氨基酸类	73-22-3

**10种植物激素绝对定性定量分析**
编号	中文名	英文名	简称
1	反-玉米素	Trans-Zeatin	t-ZT
2	赤霉素	Gibberellin A3	GA3
3	吲哚乙酸	3-Indoleacetic acid	IAA
4	水杨酸	Salicylic acid	SA
5	茉莉酸	Jasmonic acid	JA
6	脱落酸	Abscisic acid	ABA
7	吲哚丁酸	3-Indolebutyric acid	IBA
8	6-苄氨基嘌呤	6-Benzylaminopurine	6-BA
9	茉莉酸甲酯	Methyl jasmonate	JA-ME
10	激动素	6-Furfurylaminopurine	6-KT

**48种胆汁酸绝对定性定量分析**
编号	简称	常用名	编号	简称	常用名
1	DLCA	Dehydrolithocholic acid	25	3β-CA	3β-Cholic Acid
2	LCA	Lithocholic acid	26	α-MCA	α-Muricholic acid
3	ILCA	Isolithocholic acid	27	12-DHCA	12-Dehydrocholic acid
4	NDCA	23-Nordeoxycholic acid	28	λ-MCA （HCA)	λ-Muricholic acid
5	6-KLCA	6-Ketolithocholic acid	29	CA	Cholic acid
6	7-KLCA	7-Ketolithocholic acid	30	AlCA	Allocholic acid
7	12-KLCA	12-Ketolithocholic acid	31	GLCA	Glycolithocholic acid
8	ApCA	Apocholic acid	32	GCDCA	Glycochenodeoxycholic acid
9	MoCA	Murocholic acid	33	TLCA	Taurolithocholic acid
10	TCDCA-d5	Taurochenodeoxycholic-d5 acid	34	GHDCA	Glycohyodeoxycholic acid
11	β-HDCA	β-Hyodeoxycholic Acid	35	GDHCA	Glycodehydrocholic acid
12	UDCA	Ursodeoxycholic acid	36	λ-GMCA (GHCA)	Glyco-λ-muricholic acid
13	CDCA	Chenodeoxycholic acid	37	TDCA	Taurodeoxycholic acid
14	DHCA	Dehydrocholic acid	38	GUDCA	Glycoursodeoxycholic acid
15	HDCA	Hyodeoxycholic acid	39	TUDCA	Tauroursodeoxycholic acid
16	IDCA	Isodeoxycholic acid	40	THDCA	Taurohyodeoxycholic acid
17	NCA	23-Norcholic acid	41	TCA	Taurocholic acid
18	DCA	Deoxycholic acid	42	GDCA	Glycodeoxycholic acid
19	DiLCA	Dioxolithocholic acid	43	TDHCA	Taurodehydrocholic acid
20	6,7-DKLCA	6,7-Diketolithocholic acid	44	α-TMCA	Tauro-α-muricholic acid
21	7-KDCA	7-Ketodeoxycholic acid	45	β-TMCA	Tauro-β-muricholic acid
22	β-MCA	β-Muricholic acid	46	λ-TMCA	Tauro-λ-muricholic acid
23	3-DHCA	3-Dehydrocholic acid	47	ω-TMCA	Tauro-ω-muricholic acid
24	UCA	Ursocholic acid	48	GCA	Glycocholic acid

**18种辅酶绝对定性定量分析**
序号	常用名	中文名字
1	NAD+	氧化烟酰胺腺嘌呤二核苷酸
2	NADP+	氧化烟酰胺腺嘌呤二核苷酸磷酸
3	NADH	还原型烟酰胺腺嘌呤二核苷酸
4	NADPH	还原型酰胺腺嘌呤二核苷酸磷酸
5	ATP	三磷酸腺苷
6	ADP	二磷酸腺苷
7	AMP	一磷酸腺苷
8	Creatinephosphate	磷酸肌酸
9	Creatine	肌酸
10	Creatinine	肌苷
11	2'-deoxyuridine	脱氧尿苷
12	Cytosine	胞嘧啶
13	Adenosine	腺苷
14	Thymine	胸腺嘧啶
15	Cytidine	胞啶
16	Uridine	尿苷
17	Guanosine	鸟苷
18	UMP	尿苷酸

**105种抗生素绝对定性定量分析**
物质类别	物质列表
Sulfonamides （磺胺类：30种）	sulfapyridine, sulfadoxine, sulfathiazole, sulfamethizole, sulfamethazine, sulfadiazine, sulfaquinoxaline, sulfachloro pyridazine, sulfisomidine, sulfadimethoxine, sulfaguanidine, sulfanilamide, sulfacetamide, sulfamerazine, sulfamoxole, succinylsulfathiazole, sulfamethoxazole, sulfamethoxypyridazine, sulfamonomethoxine, sulfisoxazole, sulfanitran, sulfaclozine , sulfameter , trimethoprim, sulfasalazine, N-acetylsulfapyridine, N-acetylsulfadiazine, N-acetylsulfamethazine, N-acetylsulfamethoxazole, N-acetylsulfamerazine
Quinolones （喹诺酮类：20种）	enrofloxacin, ciprofloxacin, danofloxacin, difloxacin, enoxacin, lomefloxacin, norfloxacin, ofloxacin, pefloxacin, sarafloxacin, sparfloxacin, cinoxacin, moxifloxacin, flumequine, pipemidic acid, nalidixic acid, fleroxacin, oxolinic acid, piromidic acid, levofloxacin
Tetracyclines （四环素类：14种）	oxytetracycline, tetracycline, chlortetracycline, minocycline, methacycline, doxycycline, 4-epianhydrotetracycline, Isochlortetracycline, 4-epitetracycline, anhydrotetracycline, 4-epianhydrochlortetracycline, 4-epioxytetracycline, α-apo-oxytetracycline, β-apo-oxytetracycline
Macrolides （大环内酯类：16种）	erythromycin, clarithromycin, clindamycin, roxithromycin, tilmicosin, lincomycin, ormetoprim, oleandomycin, azithromycin, virginiamycin M1, virginiamycin S1, kitasamycin, spiramycin, tylosin, midecamycin, midecamycin
β-lactams （β-内酰胺类：14种）	Cefalexin, cefradine, cefotaxime, ampicillin, amoxicillin, dicloxacillin, cefazolin, cloxacillin, cefaclor, penicillin V, cefapirin, piperacillin, ceftiofur, penicillin G
Chloramphenicols （氯霉素类：3种）	chloramphenicol, thiamphenicol, florfenicol
Polypeptides and other antibiotics （多肽和其他抗生素：8种）	bacitracin A, colistin A, colistin B, furazolidone, monensin, salinomycin, narasin, tiamulin

Project advantages

a. Standardized technical platform: with standardized experimental operation specifications and advanced technical platform, short experimental period and reliable quality.

b. Advanced detection equipment: Aglient 7890A/5975C single quadrupole gas-mass spectrometer, Aglient 8890-7010B triple quadrupole gas-mass spectrometer, SCIEX TripleTOF 5600 plus high-resolution mass spectrometer, SCIEX QTRAP 4500 and SCIEX Triple Quad 4500 triple quadrupole mass spectrometry systems, and Agilent 600 M metabolomics analysis platforms such as ultra-low temperature nuclear magnetic resonance spectrometer.

c. Professional database: several commercial databases are available, while over one hundred standard sample databases have been established to make the characterization more accurate.

d. Multiple quality control guarantees: the analysis process adopts strict method validation steps to ensure reliable experiments.

e. Provide customized services: With professional bioinformatics team and large computer cluster, we can provide partners with personalized bioinformatics analysis services.

f. Complete after-sales service: perfect after-sales service, from report interpretation to article publication, can provide efficient and high-quality service throughout the whole process.

g. Diversified development types: Several major categories of common metabolites have been developed for targeted metabolomics analysis, such as metabolic pathways, targeted metabolites, plant metabolites, organic compounds, inorganic compounds, oxidative stress compounds, etc.

literature：Mathé EA, et al., Noninvasive urinary metabolomic profiling identifies diagnostic and prognostic markers in lungcancer. 2014, Cancer Research, 74(12):3259-70 (IF=9.32)

Experimental materials:
Pre screening samples (1005 cases): Spend ten years (1998-2007) Collecting samples: 469 patients (pre treatment urine)+536 healthy individuals
Post validation samples (158 cases): Recently collected samples (2008-2010): 80 patients (pre treatment urine)+78 healthy individuals
Tissue samples: 48 tumor tissues and surrounding non tumor tissues
Research process:
Overview: Early non target screening+later targeted MRM validation
Experimental process:

Research conclusion:
Pre screening: Non targeted metabolomics analysis of pre screened samples revealed a total of 1807 signals detected in positive ion mode and 1359 signals detected in negative ion mode. Through the detection of smoking related metabolites (Cotinine, Nicotine-N '- oxide, and Trans-3' - hydroxycotinine), it was found that smoking and non-smoking populations can be well distinguished, verifying the feasibility of the analysis method.

Data analysis:
a. Diagnosis and typing study: Using statistical analysis methods, after excluding interference from race and gender, the author identified four differential metabolites: NANA, Cortisol sulfonate, Creatine riboside, and 561+(unidentified substances). ROC analysis found that the AUC values of four differential metabolites ranged from 0.63 to 0.76 in all populations, and from 0.59 to 0.70 in the I-II lung cancer population. Among them, using Creatine riboside or all four metabolites for prediction is more accurate (P<0.0001).

　　Overlap between signals that are predictive of lung cancer status in the training set based on the Random Forests classification.

　　Receiver Operating Characteristic (ROC) analysis of individual metabolites and their combination

b. Prognostic study: After considering multiple factors such as gender, race, disease stage, tissue sectioning, smoking history, radiotherapy and chemotherapy, and surgery, the author found that high levels of NANA, Cortisol sulfate, Creatine riboside, and 561+resulted in poor prognosis (low survival rate), and the correlation between these four metabolites and survival rate was independent but cumulative; In the population of stage I-II lung cancer, high levels of Creatine riboside and 561+can also reduce patient survival.

　　Kaplan-Meier survival estimates in the training set are depicted for the top four predictive metabolites

c. Later validation: Using non targeted metabolomics methods, it was found that the levels of Creatine riboside, NANA, and 561+in the patient's urine were significantly increased in the validation population (80 patients+78 healthy individuals). After excluding interference from age, gender, and race, 198 samples (92 patients+106 healthy individuals) were selected for targeted metabolomics to verify the correlation between these four metabolites and diseases. In addition, to demonstrate the stability of these four metabolites, the author repeated the experiment after storing the samples for two years, demonstrating their clinical value.

　　Abundance and validation of metabolites that were top contributors in the classification of patients as lung cancer or healthy controls.

d. Extension of mechanism research: The author finally detected the content of the four metabolites mentioned above in tumor tissue and found that compared with adjacent tissues, the content of Creatine riboside and NANA in tumor tissue was significantly increased, while the content of Creatine also increased.

　　Linking urinary metabolites to lung cancer tissue metabolome.

Summary:
This article is a very classic example of metabolomic biomarker research. The early non targeted screening and later targeted validation are very valuable for reference and learning.
At the end of the article, the body fluid samples were returned to tissue samples again, opening the door to later mechanism research. This research approach is also very worthy of clinical research reference.

1. Reference standard for sample size in metabolomics

Detection strategy/method	Blood sample	Urine	Cerebrospinal fluid	Feces	Microorganisms	Cells	Organization	Plants
Non-targeted metabolome	500ul	1ml	200ul	2g	250mg	10⁷个	250mg	250mg
Targeted metabolome	500ul	1ml	200ul	2g	250mg	10⁷个	250mg	250mg
NMR metabolome	500ul	1ml	500ul	2g	500mg	10⁷个	500mg	500mg

2. Reproducibility requirements for metabolomics delivery biology

　　The differential variation in the metabolome is a presentation of the amplification effect of genomic changes, and the volatility of metabolites in different individuals is then greater. On the other hand, differential analysis in metabolomics is generally based on multivariate statistical analysis methods such as PCA and PLS-DA, and only the principal components sampled from larger sample sizes are representative of the population. Therefore, metabolomics requires more biological replicates of samples than technical methods such as genomics.

　　a. Clinical specimens greater than 30 cases;

　　b. greater than 10 cases for model animal samples;

　　c. greater than 8 cases per group for plant microbial samples;

　　d. Cellular samples greater than 6.

3. Metabolomics send sample collection criteria

Type of sample		Collection criteria
Blood	Serum	3-4mL of blood is drawn intravenously and placed in a blood collection tube (without any anticoagulant or procoagulant); left at 4°C for 30-60min (no more than 2h) to allow the blood to clot; centrifuged at 3500rpm, 4°C for 10 minutes, and after the blood cells have settled completely, the supernatant is aspirated and stored at -80°C.
	Plasma	The blood sample is collected and placed in a collection tube containing an appropriate amount of anticoagulant (Citrate anticoagulant is recommended; Heprin anticoagulant is recommended for NMR; EDTA anticoagulant (K2, K3, Na, absolutely no Li) is recommended for mass spectrometry; the tube is repeatedly inverted to mix the anticoagulant with the blood; the blood sample is centrifuged at 2000-3000 rpm for 10 min (centrifugation is performed within 1 h at room temperature after collection; blood samples can be processed within 4 h on ice after collection) and the supernatant is plasma; it is stored in a refrigerator at -80°C.
Urine		The urine (no bacterial inhibitors such as sodium azide should be added to avoid interference with the mass spectrometry test) is collected in a centrifuge tube (PP material recommended) and centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant is removed and stored at -80°C. It is recommended that urine creatinine is measured at the same time as the metabolomics test.
Other body fluid samples		The collection process is carried out without the addition of any other reagents, and the tubes are then frozen in liquid nitrogen and stored at -80°C.
Cells	Adherent cells (not less than 1.0E7 cells per dish)	Before treatment, count the cells in each dish, discard the culture medium, add 10 ml of deionised water to each dish to wash off the medium, shake for 2 s (not too vigorously to prevent cell breakage), add 15 ml of liquid nitrogen to quench (2mins), add 1.5 ml of methanol pre-chilled at -80°C and scrape with a spatula.
	Cells that do not adhere to the wall	The suspension was transferred intact to a 1.5 ml centrifuge tube and centrifuged at 4 °C for 10 min at 13000 rpm. A fixed volume of supernatant (equal volume per dish, e.g. 0.8 mL) was taken and placed in a 1.5 mL centrifuge tube and stored at -80 °C. At the same time, the cell debris was weighed and recorded.
	Cell culture solution (2mL/case)	A fixed volume of cell culture solution was aspirated from each dish and placed in a 2mL centrifuge tube at 10,000rpm for 10 minutes at 4°C. The supernatant was removed and stored at -80°C.
Feces		Three spots were picked from different parts of the faeces, weighed and placed in centrifuge tubes (the addition of antibacterial agents such as sodium azide is strictly forbidden to avoid interference with the mass spectrometry test) and stored at -80°C.
Organization	Animal tissue samples	Operate on ice throughout the procedure to minimise handling time. Remove blood (heart, liver, kidney and lungs can be perfused, other tissues can be washed several times with pre-cooled PBS) and store in liquid nitrogen at -80°C.
	Human tissue samples and tumour samples	Minimise handling time. The blood is removed (pre-cooled PBS dripping several times), disturbed, snap frozen in liquid nitrogen and stored at -80°C.
Microbiological and fungal samples		Collect 10⁷ bacteria/case or 500mg/case of mycelium in a centrifuge tube, if the exact amount of mycelium cannot be judged, it can be the size of a soybean grain, rapidly snap frozen in liquid nitrogen and stored at -80°C.
Plant samples		It is recommended that crushed samples are weighed, snap frozen in liquid nitrogen and stored at -80°C. Uncrushed samples can also be sent.