Leymus chinensis was grown at Inner Mongolia Agricultural University field experimental site in Chifen(41°17′10″～45°24′15″ N，116°21′07″～120°58′52″E),Inner Mongolia, China, in 2018.We are licensed by the local agriculture and pastoral bureau and certified by grass industry experts, and we are conducting experiments in accordance with the local grassland protection law. The selected Leymus chinensis is naturally grown on native grassland. When taking samples of Leymus chinensis, the staff followed the local grassland protection law and the regulations on sampling wild species in the convention on trade in endangered species of wild fauna and do as the :https://www.cites.org/ requested. The identification of Leymus chinensis samples was conducted after consulting with Mr Zhang weiguo, a well-known expert in plant classification in China. Zhang weiguo devoted himself to the identification and classification of forage grass species in the field all the year around, with rich field work experience and scientific research foundation.We took Leymus chinensis as the experimental material and started the experiment on August 20, 2017. When the forage was harvested, the growth periods were jointing stage and solid stage. The jointing stage is a period which has a rapid growth of stem on Leymus chinensis. Generally, the first stem node of more than 50% plants in the whole field is exposed to the ground by 1.5～2.5cm, and solid stage refers to the period from flowering to maturity of forage seeds. We took samples with sterilized gloves and cut them with sterilized scissors to gained two stage samples . Each sample was 200g, and one sample was placed in an envelope bag, then the samples were dried in a 105℃ oven for 30 min. Washing the Leymus chinensis samples with PBS buffer, wraping them with tin foil, marking them and immediately freeze them in liquid nitrogen, and then transfer them to -80℃ refrigerator for later use.
The contents of crude protein (CP) was tested in accordance with the reference of the AOAC international . The content of neutral detergent fiber (NDF) and acid detergent fiber (ADF) were analyzed according to the Ankom A200 fiber analyzer (Ankom Technology, Macedon, NY) by the method of Van Soest.
80 mg samples were added to liquid nitrogen for grinding, and 1 ml of methanol/acetonitrile/water (2:2:1, v/v) was poured into the mixture. Then, the samples were rotated and mixed well. After ultrasonic crushing at low temperature for 0.5 h, the samples were repeated three times.
Liquid mass spectrometry
In order to obtain reliable data, this study added QC sample analysis to monitor the entire experimental process. The metabolites of samples were extracted by precipitation protein method, and the same amount of samples were mixed to prepare quality control (QC) samples. The samples were randomly sorted by computer, and QC samples were inserted before.
Description of metabolite extraction
Samples were defrosted on ice and metabolites were extracted with 50% methanol buffer. Temporarily shelved, 20 ml samples were pre-cooled with 120 ml 50% methanol, vortex for 1 min, incubate at room temperature for 10 min. Set -20°C for 24 h. After centrifugation for 20 min, the supernatant was transferred to the new 96-well plate. Pre-storage qualitative analysis at -80°C.
Description of liquid phase parameters
All samples were collected by using instructions on the LC-MS/MS system. All the chromatograms were collected by UPLC system (SCIEX, UK). ACQUITY UPLC T3 column (100 mm x 2.1 mm, 1.8 m (including waters, UK) is mainly responsible for the phase separation. Gradient elution specification was set as follows: 0～0.5 min, 5% B; 0.5～7 min, 5% to 100% B; 7～8 min, 100% B; 8～8.1 min, 100% to 5% B; 8.1 ～10 min, 5% b.
Mass spectrum parameter description
Metabolites were analyzed by a high resolution tandem mass spectrometer TripleTOF5600plus (SCIEX, UK). Q-TOF works in positive and negative ion modes. Curtain gas is 30 PSI, ion source gas1 is 60 PSI, ion source gas2 is 60 PSI, and the temperature of an interface heater is close to 650℃. For positive ion mode, the floating adjustment of ion spray voltage is 5000 V. For negative ion mode, the floating adjustment of ion spray voltage is -4500v. The mass spectrometry data were collected using IDA mode. The TOF quality range is 60 ~ 1200 Da. Scanning can be obtained in a time of 150 ms, when more than 100 counts per second (count /s), with a 1+ charge quantity, up to 12 ions can be collected. The total cycle time was set at 0.56s.
Information analysis process
In order to obtain the accuracy of data, it is necessary to analyze the offline data strictly according to the steps of information analysis. Through MSConvert software of Proteowizard, plain data is parsed and converted into readable data mzXML. Peak value is extracted by XCMS software and quality control is carried out on peak value. The substances after quality control were annotated by CAMERA and ion, and were identified by metaX software. The first level and second level information of mass spectrum were identified and matched with in-house standard product database. Metabolites were annotated through HMDB, KEGG and other databases to clarify the physicochemical properties, biological functions and characteristics of metabolites. Differential metabolites were quantitatively screened by metaX software.
Information analysis description
Peak extraction, peak grouping, correction, secondary peak grouping, isotope and adder annotation and other preprocessing of MS data were carried out by XCMS software. The raw data is converted to mzXML format and processed by XCMS. Retention time (RT) and molecular data were fused, and each ion was identified. Different peak strengths were recorded to generate a three-dimensional matrix composed of peak index, sample name and ion strength information. The exact molecular weight (m/z) and molecular weight data of samples were accurately compared through online KEGG and HMDB databases, and metabolites were annotated. If the observed value is compared with the database value and the quality difference is less than 10ppm, then the standard metabolite. In addition, we can identify and verify metabolites through an internal metabolite fragment library. The peak strength was pretreated by METAX. After eliminating the features detected in QC samples less than 1/2 or biological samples 4/5, k-nearest neighbor algorithm was used to inject the residual peak of the missing value, so as to further improve the data quality. The evaluation of PCA outlier detection and batch processing were performed on the data was obtained by preprocessing. Based on the robust loess signal correction method to control the quality is applied to the data of injection sequence, so as to weaken the change of signal strength and enhance the reliability of the experiment.
Metabolites of Leymus chinensis at different harvest times in typical grassland were analyzed by high performance liquid chromatography-mass spectrometry (HILIC UPLC-Q-TOF/MS). The analysis method is stable and the metabolomics data obtained are reliable. In the process of sample analysis, in order to verify the performance of the system, representative samples are processed after averaging by collecting QC samples. The analysis process includes all samples. QC samples are treated with actual samples and each 5 samples are added to ESI positive or negative to analyze the stability of the monitoring instrument in different batches. The similarity of QC includes peak shape, separation degree, retention time and intensity distribution of metabolites involved in configuration files. It will get the QC samples to analysis 5 times in total ion to flow chart after comparing spectral overlap, as shown in figure 5, the results show that the response intensity and retention time of chromatographic peak of basic overlap, the method is stable and reliable in the whole experiment process, good repeatability and stability , and it does not exist the technical error caused by man-made factors, the sample is suitable for this study.
We can see that the equipment of this experiment is stable, the data is reliable, and the stability is good from the figure 6. The metabolic spectrum differences obtained in the experiment can well reflect the biological differences between samples.
Leymus chinensis in typical steppe was regarded as research materials, based on metabonomics analysis, using high performance liquid chromatography tandem high-resolution mass spectrometer Triple TOF 5600 in positive and negative ions to metabolic group detection mode, combined with biological information analysis mass spectrometry data interpretation of biological information analysis mainly using XCMS software substance detection using METAX software for quantitative differences in material selection, respectively using METAX software for material level of mass spectrogram Using the in - house atlas for material secondary metabolites annotation mass spectrogram, to study the the internal causes of nutrition in Leymus chinensis at different harvest stage from the molecular level and provide a reasonable and efficient theoretical basis, to gained high quality forage production in the future research and provide the new direction and new ideas.