基于高分辨质谱和代谢组学技术评估和优化蜂王浆代谢物提取方法

doi:10.3864/j.issn.0578-1752.2020.18.017

Abstract

Abstract:

【Objective】Royal jelly, a natural product with health-promoting effect, is rich in small-molecule bioactive compounds. The systematic analysis of the metabolite extraction from royal jelly is still lacking. This study performed metabolic profiling of royal jelly with an aim to compare the metabolite extraction efficiency using different solvents, optimize the metabolite extraction protocol, and globally identify the small-molecule compounds in royal jelly. 【Method】Metabolites in royal jelly were extracted separately using six different solvents including 50% and 80% methanol, ethanol, and acetonitrile. They were analyzed by reverse phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) each combined with Quadrupole-Exactive Orbitrap high resolution mass spectrometry (HRMS). The amount and relative standard deviation (RSD) values of metabolite features among the solvent groups were compared. Principal component analysis (PCA) was conducted on the basis of these metabolite features. The compound identification was based on mass spectrometry databases and was validated with standards. Both the RSD value comparison and clustering heatmap analysis were performed for the identified compounds. Significantly differential metabolites among the solvent groups were screened out by orthogonal partial least squares-discriminant analysis (OPLS-DA), univariate analysis (Student’s t-test) and fold change (FC). 【Result】Highly polar metabolites, including two isomeric sugars (glucose and fructose), and medium and weakly polar metabolites such as lipids were sufficiently separated using the HILIC and PRLC approach, respectively. The combination of the two separation methods enabled comprehensive detection of metabolites with different polarity in royal jelly. A total of 70 high-abundance compounds were identified, including carbohydrates, amino acids, lipids, and vitamins, with the highest difference of 8 340 times in abundance. Among them, 17 compounds were reported for the first time. The lowest number of metabolite features was obtained with acetonitrile, and a worse coverage was obtained with 80% than 50% acetonitrile. For methanol and ethanol, the number of metabolite features was higher at higher solvent concentrations. The RSD values of most metabolite features were lower than 20% in all solvent groups, and the 80% acetonitrile group had the lowest proportion within 10% RSD values compared with other solvents. The RSD values of the 70 identified compounds provided further evidence for the poor repeatability of 80% acetonitrile. The PCA score plots indicated a similar metabolic profiling from the same solvent. Differences in metabolic profiling were observed among the solvents, and the largest difference existed between 80% acetonitrile and the five other solvents. The clustering heatmap showed a lower abundance of medium and weakly polar metabolites obtained from 50% solvents and a lower abundance of highly polar metabolites including, in particular, fructose, glucose, sucrose, lysine, adenosine, choline, phosphorylcholine, and gluconic acid from 80% acetonitrile. 【Conclusion】The combination of RPLC-HRMS and HILIC-HRMS enables comprehensive and accurate detection of small-molecule compounds in royal jelly. 80% methanol or 80% ethanol is an optimal solvent for metabolite extraction from royal jelly.

Key words: royal jelly, metabolomics, sample extraction, high resolution mass spectrometry (HRMS)

ZHANG LiCui,MA Chuan,FENG Mao,LI JianKe. Evaluation and Optimization of Metabolite Extraction Protocols for Royal Jelly by High Resolution Mass Spectrometry and Metabolomics[J].Scientia Agricultura Sinica, 2020, 53(18): 3833-3845.

Figures/Tables 7

Fig. 1

Fig. 2

Table 1

Tentatively identified compounds in royal jelly"

编号 No.	化合物 Compound	分子式 Molecular formula	保留时间Retention time (min)	分子量测量值Measured mass	分子量理论值Theoretical mass	质量偏差 Mass error (×10^-6)	匹配度得分Match score	平均峰面积 Average peak area	色谱柱 Column
1	马尿酸Hippuric acid^ab	C₉H₉NO₃	3.34	179.05835	179.05824	0.61	<50.0	1.93E+05	RPLC
2	咖啡碱Caffeic acid^a	C₉H₈O₄	3.65	180.04238	180.04226	0.67	85.0	3.28E+05	RPLC
3	3,10-二羟基癸酸3,10-Dihydroxydecanoic acid	C₁₀H₂₀O₄	3.94	204.13618	204.13616	0.10	<50.0	2.24E+08	RPLC
4	辛二酸Suberic acid^a	C₈H₁₄O₄	3.95	174.08918	174.08921	-0.17	97.1	1.03E+06	RPLC
5	8-羟基辛酸8-Hydroxyoctanoic acid^a	C₈H₁₆O₃	4.05	160.10992	160.10994	-0.12	<50.0	2.26E+06	RPLC
6	壬酸Nonanoic acid	C₉H₁₈O₂	4.67	158.13068	158.13068	0.00	88.1	4.79E+06	RPLC
7	2-癸二酸2-Decenedioic acid	C₁₀H₁₆O₄	4.80	200.10483	200.10486	-0.15	97.0	2.42E+08	RPLC
8	癸二酸Decenedioic acid^a	C₁₀H₁₈O₄	4.95	202.12048	202.12051	-0.15	85.2	3.83E+08	RPLC
9	10-羟基二癸烯酸10-Hydroxy-2-decenoic acid^a	C₁₀H₁₈O₃	4.96	186.12558	186.12559	-0.05	97.0	5.35E+07	RPLC
10	10-羟基癸酸10-Hydroxydecanoic acid^a	C₁₀H₂₀O₃	5.12	188.14120	188.14124	-0.21	89.0	1.90E+07	RPLC
11	十四烷二酸Tetradecanedioic acid^b	C₁₄H₂₆O₄	5.43	258.18337	258.18311	1.01	52.0	9.89E+05	RPLC
12	愈伤酸Traumatic acid^a	C₁₂H₂₀O₄	5.72	228.13631	228.13616	0.66	84.7	2.22E+07	RPLC
13	3-羟基癸酸3-Hydroxydecanoic acid^a	C₁₀H₂₀O₃	5.74	188.14121	188.14124	-0.16	89.7	8.69E+07	RPLC
14	十二烷二酸Dodecanedioic acid^a	C₁₂H₂₂O₄	5.85	230.15179	230.15181	-0.09	87.2	3.73E+07	RPLC
15	脂肪酸（12:1）FA (12:1)	C₁₂H₂₂O₂	5.91	198.16194	198.16198	-0.20	50.8	1.37E+07	RPLC
16	11-羟基十二烷酸 11-Hydroxydodecanoic acid^a	C₁₂H₂₄O₃	5.92	216.17257	216.17254	0.14	78.3	1.24E+06	RPLC
17	12-羟基十二烷酸 12-Hydroxydodecanoic acid^a	C₁₂H₂₄O₃	6.05	216.17256	216.17254	0.09	66.8	1.48E+06	RPLC
18	柯因Chrysin^a	C₁₅H₁₀O₄	6.51	254.05777	254.05791	-0.55	97.1	2.12E+06	RPLC
19	脂肪酸（14:2）FA (14:2)	C₁₄H₂₄O₂	6.61	224.17759	224.17763	-0.18	89.4	2.39E+06	RPLC
20	13-羟基十四烷酸13-Hydroxytetradecanoic acid	C₁₄H₂₈O₃	6.74	244.20393	244.20384	0.37	95.2	7.39E+06	RPLC
21	脂肪酸（16:1）FA (16:1)	C₁₆H₃₀O₂	7.48	254.22445	254.22458	-0.51	95.2	5.32E+05	RPLC
22	烟酰胺Nicotinamide^a	C₆H₆N₂O	1.92	122.04801	122.04801	0.00	81.7	1.34E+06	HILIC
23	吡哆醛Pyridoxal	C₈H₉NO₃	2.25	167.05826	167.05824	0.12	88.5	3.04E+06	HILIC
24	泛酸Pantothenic acid^a	C₉H₁₇NO₅	3.26	219.11061	219.11067	-0.27	80.7	5.03E+07	HILIC
25	乙酰胆碱Acetylcholine^a	C₇H₁₅NO₂	3.28	145.11018	145.11028	-0.69	95.8	1.61E+09	HILIC
26	琥珀酸Succinic acid^a	C₄H₆O₄	3.39	118.02665	118.02661	0.34	83.7	4.34E+07	HILIC
27	烟酸Nicotinic acid^a	C₆H₅NO₂	3.98	123.03209	123.03203	0.49	64.7	8.27E+05	HILIC
28	2′-脱氧腺苷2′-Deoxyadenosine^ab	C₁₀H₁₃N₅O₃	4.01	251.10167	251.10184	-0.68	<50.0	9.55E+05	HILIC
29	腺嘌呤Adenine^a	C₅H₅N₅	4.87	135.05452	135.05450	0.15	90.8	2.03E+08	HILIC
30	尿苷Uridine^a	C₉H₁₂N₂O₆	5.42	244.06970	244.06954	0.66	84.7	2.53E+07	HILIC
31	腺苷Adenosine^a	C₁₀H₁₃N₅O₄	5.54	267.09670	267.09675	-0.19	97.4	2.74E+08	HILIC
32	肌酐Creatinine^a	C₄H₇N₃O	5.58	113.05896	113.05891	0.44	<50.0	4.75E+05	HILIC
33	胞嘧啶Cytosine^a	C₄H₅N₃O	5.94	111.04327	111.04326	0.09	95.5	7.44E+05	HILIC
34	胆碱Choline^a	C₅H₁₃NO	6.09	103.09963	103.09971	-0.78	91.3	2.32E+08	HILIC
35	溶血磷脂酰胆碱（18:3）LysoPC (18:3)^b	C₂₆H₄₈NO₇P	6.32	517.31677	517.31684	-0.14	78.6	2.04E+06	HILIC
36	N,N-二乙基乙醇胺N,N-Diethylethanolamine^b	C₆H₁₅NO	6.33	117.11532	117.11536	-0.34	75.7	4.21E+07	HILIC
37	N-乙酰基组胺N-Acetylhistamine^ab	C₇H₁₁N₃O	6.60	153.09019	153.09021	-0.13	91.4	4.34E+06	HILIC
38	肌苷Inosine^a	C₁₀H₁₂N₄O₅	6.76	268.08081	268.08077	0.15	90.0	5.05E+06	HILIC
39	次黄嘌呤Hypoxanthine^a	C₅H₄N₄O	6.76	136.03854	136.03851	0.22	92.8	4.34E+06	HILIC
40	鸟嘌呤Guanine^a	C₅H₅N₅O	6.93	151.04944	151.04941	0.20	93.0	2.70E+05	HILIC
41	甜菜碱Betaine^a	C₅H₁₁NO₂	7.26	117.07893	117.07898	-0.43	96.0	2.18E+07	HILIC
42	鸟苷Guanosine^a	C₁₀H₁₃N₅O₅	7.27	283.09147	283.09167	-0.71	98.9	1.32E+06	HILIC
43	果糖Fructose^a	C₆H₁₂O₆	7.35	180.06350	180.06339	0.61	92.4	4.07E+08	HILIC
44	胡芦巴碱Trigonelline^a	C₇H₇NO₂	7.39	137.04766	137.04768	-0.15	93.8	3.81E+07	HILIC
45	胆碱Proline^a	C₅H₉NO₂	7.61	115.06330	115.06333	-0.26	97.4	6.82E+07	HILIC
46	肉碱Carnitine^ab	C₇H₁₅NO₃	7.66	161.10514	161.10519	-0.31	82.4	1.26E+06	HILIC
47	γ-氨基丁酸γ-Aminobutyric acid^a	C₄H₉NO₂	7.66	103.06332	103.06333	-0.10	92.1	2.88E+06	HILIC
48	牛磺酸Taurine^a	C₂H₇NO₃S	7.66	125.01462	125.01466	-0.32	82.2	1.53E+06	HILIC
49	葡萄糖Glucose^a	C₆H₁₂O₆	7.67	180.06344	180.06339	0.28	97.8	1.30E+08	HILIC
50	吡哆胺Pyridoxamine	C₈H₁₂N₂O₂	7.81	168.08986	168.08988	-0.12	87.4	4.25E+06	HILIC
51	β-丙氨酸β-Alanine^a	C₃H₇NO₂	7.88	89.04772	89.04768	0.45	88.9	5.48E+06	HILIC
52	苏氨酸Threonic acid^a	C₄H₈O₅	7.99	136.03725	136.03717	0.59	55.0	7.53E+05	HILIC
53	蔗糖Sucrose^a	C₁₂H₂₂O₁₁	8.08	342.11652	342.11621	0.91	93.6	1.71E+08	HILIC
54	葡萄糖酸Gluconic acid^a	C₆H₁₂O₇	8.24	196.05822	196.05830	-0.41	95.9	4.08E+08	HILIC
55	谷氨酸Glutamic acid^a	C₅H₉NO₄	8.26	147.05315	147.05316	-0.07	95.2	7.14E+06	HILIC
56	甘油3-磷酸乙醇胺 Glycerol 3-phosphoethanolamine^b	C₅H₁₄NO₆P	8.29	215.05582	215.05587	-0.23	<50.0	2.47E+06	HILIC
57	N3,N4-二甲基-L-精氨酸N3,N4-Dimethyl-L-arginine^b	C₈H₁₈N₄O₂	8.37	202.14296	202.14298	-0.10	90.5	8.42E+06	HILIC
58	5′-单磷酸腺苷 Adenosine 5′-monophosphate^a	C₁₀H₁₄N₅O₇P	8.45	347.06309	347.06308	0.03	84.3	1.36E+07	HILIC
59	尿苷一磷酸Uridine monophosphate^ab	C₉H₁₃N₂O₉P	8.52	324.03609	324.03587	0.68	86.2	8.36E+05	HILIC
60	N6,N6,N6-三甲基-L-赖氨酸N6,N6,N6-Trimethyl-L-lysine^b	C₉H₂₀N₂O₂	8.53	188.15221	188.15248	-1.44	56.5	5.95E+06	HILIC
61	天冬氨酸Aspartic acid^a	C₄H₇NO₄	8.54	133.03753	133.03751	0.15	90.5	1.72E+06	HILIC
62	N6-甲基-L-赖氨酸N6-Methyl-L-lysine^b	C₇H₁₆N₂O₂	8.59	160.12116	160.12118	-0.12	87.6	1.44E+06	HILIC
63	1-甲基组氨酸1-Methylhistidine^ab	C₇H₁₁N₃O₂	8.71	169.08506	169.08513	-0.41	91.2	2.11E+06	HILIC
64	组氨酸Histidine^a	C₆H₉N₃O₂	8.73	155.06952	155.06948	0.26	98.3	1.36E+07	HILIC
65	赖氨酸Lysine^a	C₆H₁₄N₂O₂	8.81	146.10548	146.10553	-0.34	90.2	7.32E+07	HILIC
66	鸟氨酸Ornithine^a	C₅H₁₂N₂O₂	8.86	132.08986	132.08988	-0.15	92.8	8.47E+05	HILIC
67	鸟苷单磷酸Guanosine monophosphate^ab	C₁₀H₁₄N₅O₈P	8.87	363.05817	363.05800	0.47	78.1	2.64E+06	HILIC
68	烟酰胺腺嘌呤二核苷酸 Nicotinamide adenine dinucleotide^ab	C₂₁H₂₇N₇O₁₄P₂	8.94	663.10950	663.10912	0.57	94.2	4.80E+05	HILIC
69	UDP-N-乙酰氨基葡萄糖UDP-N-Acetylglucosamine^b	C₁₇H₂₇N₃O₁₇P₂	9.08	607.08223	607.08157	1.09	91.0	1.29E+06	HILIC
70	磷酸胆碱Phosphorylcholine^ab	C₅H₁₄NO₄P	9.26	183.06600	183.06604	-0.22	97.5	7.18E+07	HILIC

Table 1

Fig. 3

Fig. 4

Fig. 5

Fig. 6

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Evaluation and Optimization of Metabolite Extraction Protocols for Royal Jelly by High Resolution Mass Spectrometry and Metabolomics

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