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Acta Agronomica Sinica ›› 2019, Vol. 45 ›› Issue (12): 1899-1904.doi: 10.3724/SP.J.1006.2019.91017

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Analysis method of wheat germination metabolomics based on UPLC-QTOF/ MS

Li-Na WANG,Bu-Jun WANG()   

  1. Institute of Crop Sciences, Chinese Academy of Agricultural Sciences / Cereal Quality Supervision and Testing Center, Ministry of Agriculture, Beijing 100081, China
  • Received:2019-02-25 Accepted:2019-06-24 Online:2019-07-19 Published:2019-07-19
  • Contact: Bu-Jun WANG E-mail:wangbujun@caas.cn
  • Supported by:
    This study was supported by the National Grain and Oil Crop Product Quality and Safety Risk Assessment Special(GJFP2018001);the Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences;the National Key Research and Development Program(2016YFF0201803)

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Abstract:

To investigate the effects of different extraction methods on the extraction of wheat metabolites in germination, we established a pre-treatment and analysis method for the metabolomics samples based on UPLC-QTOF/MS. In this study, we used Zhoumai 26 grains germinated for 2 days, and designed L9(3 4) orthogonal experiments with extraction solvents, extraction methods and extraction time three levels based on UPLC-QTOF/MS. Besides, the best combination of extraction was obtained by principal component and cluster analysis: 1609 metabolic peaks were detected by 80% acetonitrile (0.1% formic acid) with shocking for 30 min, indicating that the extraction solvent plays a major role in the extraction of metabolites. A total of 92 wheat metabolites were identified in this study.

Key words: metabolomics, wheat, UPLC-QTOF/MS, extraction method, extraction solvent, orthogonal experimental

Table 1

Orthogonal test factors and levels"

水平
Level		 因素Factor
提取溶剂
Extraction solvent		 提取方式
Extraction method		 提取时间
Extraction time (min)
1 纯水(0.1%甲酸) Water (0.1%FA) 振摇 Shock 10
2 80%甲醇(0.1%甲酸) 80% methanol (0.1%FA) 超声 Ultrasound 15
3 80%乙腈(0.1%甲酸) 80% acetonitrile (0.1%FA) 静置 Stand still 30

Table 2

Orthogonal test results"

组号
Group		 试验号
Experiment number		 提取溶剂
Extraction solvent		 提取方式
Extraction method		 提取时间
Extraction time		 提取峰
Extraction peak
A 1 1 1 1 1134
2 1 2 2 1097
3 1 3 3 1069
B 4 2 3 1 1269
5 2 1 2 1448
6 2 2 3 1257
C 7 3 2 1 1275
8 3 3 2 1359
9 3 1 3 1609
K1 3300 4191 3678
K2 3974 3629 3904
K3 4243 3697 3935
R 943 562 257

Supplementary Fig. 1

Total ion current map in positive ion mode of QC sample (five replicates)"

Fig. 1

Effect of single factor on peak extraction"

Fig. 2

Different processing PCA analysis"

Supplementary Fig. 2

HCA analysis of different treatment abundances of identified metabolites"

Supplementary Table 1

Detection of identified metabolites in different treatments"

代谢物名称
Metabolite name		 分类
Classification		 相对分子质量
Relative mass (m z-1)		 保留时间
Retention time (min)		 1 2 3 4 5 6 7 8 9
Phenethylamine Amines 122.1 0.044 + + + + + + + + +
Adenine Imidazopyrimidines 136.1 0.154 + + + + + + + + +
Mesoxalate Organic acid 136 0.168 + + + + + + + + +
Aniline Amines 111.1 0.25 + + +
Methionine Amino acids 299.1 0.257 + + + + + +
2,5-Dimethylpyrazine Pyrazines 109.1 0.257 + +
1,3-Diaminopropane Polyamine 97.08 0.271 + +
PCe 36:2 Lipids 772.6 0.305 + +
PCe 38:5 Lipids 794.6 0.388 + +
DG(18:0/20:4/0:0)[iso2] Lipids 639.5 0.395 + + + + + + +
TG 54:6 (18:2/18:2/18:2) Lipids 901.7 0.401 + + + +
3-Ureidopropionate Amino acids 265.1 0.408 + + + +
L-Cysteic acid Amino acids 339 0.45 + +
Pyruvic aldehyde Aldehydes 145 0.505 + + + + + + + +
cAMP Nucleotides 347.1 0.505 + +
3-Nitro-L-Tyrosine Amino acids 249 0.511 + + + + +
O-Phospho-L-Serine Lipids 203 0.511 + + +
Methylmalonate Organic acid 136.1 0.518 + + + + + + + +
Alpha-Hydroxyisobutyric Acid Organic acid 127 0.518 + + + + + + + +
Leucine Amino acids 170.1 0.518 + + +
2-Hydroxy-4-(Methylthio)Butyric Acid Amino acids 173 0.518 +
L-2,3-Diaminopropionic Acid Amino acids 143 0.518 + +
Melibiose Carbohydrates 360.2 0.539 + + + +
D-(+)-Raffinose Carbohydrates 505.2 0.546 + + + + + + +
L-Norvaline Amino acids 118.1 0.56 + + + + + + + + +
3-Hydroxy-3-Methylglutarate Organic acid 325.1 0.566 + + + + + + + + +
Tyrosine Amino acids 182.1 0.573 + + + + + +
1-Naphthylamine Amines 144.1 0.573 + + + + + + + +
Methyl Acetoacetate Organic acid 117.1 0.58 + +
(R)-Malate Organic acid 152.1 0.58 + + + + + +
Indole Indoles 118.1 0.58 + + + + + + + + +
Tryptophan Amino acids 205.1 0.587 + + + + + + + + +
1-Aminocyclopropane-1-Carboxylate Amino acids 124 0.594 + + + + + +
Dihydroxyfumaric Acid Organic acid 187 0.608 + +
4-Guanidinobutanoate Amines 146.1 0.608 + + + + + + + +
Ascorbate Vitamins 199 0.608 + + +
Uridine 5'-Diphospho-N-Acetylgalactosamine Amino Sugars 608.1 0.621 + + +
Prenol Fatty alcohols 104.1 0.642 + + + +
3-Hydroxyanthranilate Organic acid 307.1 0.656 +
Betaine Amines 235.2 0.67 +
Trehalose Carbohydrates 360.2 0.676 + +
D-Fructose 6-Phosphate Carbohydrates 261 0.704 + + +
Succinic Acid Organic acid 136.1 0.731 + + + + +
Glycolaldehyde Dimer Aldehydes 98.98 0.731 + + + + + + +
Adenosine Nucleosides 268.1 1.669 + + + +
S-Carboxymethyl-L-Cysteine Amino acid 218 1.752 + + + + + +
2'-Deoxyguanosine Nucleotides 268.1 2.064 + + + + +
PEp 40:4 Lipids 797.6 2.586 + +
Phenylalanine Amino acids 188.1 2.855 + + + + + + + + +
C20DH Sphingomyelin Lipids 761.7 3.125 +
Riboflavin Vitamins 377.1 3.27 +
19:0 Cholesteryl ester Lipids 689.6 3.655 + +
PC 36:5-A Lipids 797.6 3.862 + +
Cholesteryl Acetate Lipids 857.7 4.655 + +
PC 36:2 Lipids 803.6 4.834 + +
Tricosanoic Acid Fatty acids 393.3 6.056 + +
Desmosterol Lipids 385.3 6.812 + +
Hexadecanoic Acid Lipids 274.3 6.977 + + + + + + +
Myristic Acid Lipids 251.2 7.22 + + + +
Heptadecanoic Acid Lipids 288.3 7.661 + + + + + + +
Lysine Amino acids 293.2 8.729 + +
Acetoin Ketones 177.1 8.915 + + + + + + +
Sphinganine Amines 302.3 8.949 + + + + + +
Valine Amino acids 235.2 9.928 + + +
MG (16:0/0:0/0:0) Lipids 353.3 10.21 + + + + + +
LPE 16:0 Lipids 454.3 10.68 + +
LPC 18:1 Lipids 522.4 11.19 + + + + +
Guanine Imidazopyrimidines 152.1 11.21 + +
Docosanoic Acid Fatty acyls 358.4 11.8 + + + + + + +
13Z-Docosenoic Acid Lipids 356.4 12.04 + + +
LPC 18:0 Lipids 524.4 12.6 + + + +
LPC 20:1 Lipids 550.4 12.92 + +
N-Palmitoyl-Sphingosine Lipids 560.5 14.38 + + +
Alpha-Tocopherol Vitamins 431.4 14.53 + + + +
Deoxycorticosterone Acetate Lipids 395.2 14.66 + + + + +
Hexadecanol Fatty alcohols 265.3 14.76 + + + +
Leukotriene B4 Lipids 337.2 15.22 + + + + + + + + +
N-(Octadecanoyl)-Dihydroceramide Lipids 568.6 15.25 + + + + + + + +
N-Acetyl-Dl-Glutamic Acid Amino acids 207.1 15.3 + + + + +
LPE 20:5 Lipids 517.3 15.51 + + +
3-(2-Hydroxyphenyl)Propanoate Organic acid 167.1 15.69 + + + +
Bis(2-Ethylhexyl)Phthalate Organic acid 391.3 15.7 + + + + + + + + +
PC 34:3 Lipids 778.5 15.92 + +
Azelaic acid Organic acid 189.1 16.8 + + + + +
PC 32:0 Lipids 756.6 17.25 + + +
PE 40:5 Lipids 794.6 17.64 + +
1,2-Dipalmitoyl-Sn-Glycerol Lipids 591.5 17.73 + + + + + +
9Z-Hexadecenoic Acid Lipids 277.2 17.88 + + + + + +
PC 34:0 Lipids 784.6 18.4 + + + + + +
PE 34:1 Lipids 740.5 18.44 + + + + + +
PE 34:2 Lipids 716.5 18.55 + + +
4-Aminobutanoate Organic acid 207.1 18.66 + + +
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