The content of endogenous GAs, IAAs and CTK in the young stem near the growing point was determined using 6 dps samples from both varieties. Plant samples were frozen in liquid nitrogen and ground into powder. Fifty mg of sample powder was weighted and dissolved in 1 mL methanol/water/formic acid (15:4:1, v/v/v). 10 μL internal standard mixed solution (100 ng/mL) was added into the extract as internal standards for the quantication. The mixture was vortexed for 10 min, then centrifuged for 5 min (12,000 r/min, 4 °C). The supernatant was evaporated to dryness and dissolved in 100 μL 80% methanol (v/v) and filtered with 0.22 μm membrane filter for further LC-MS/MS analysis [60 (link),61 (link)]. The extracts were analyzed using an UPLC-ESI-MS/MS system (UPLC, ExionLC™AD, https://sciex.com.cn/ accessed on 5 October 2021); MS, Applied Biosystems 6500 Triple Quadrupole, according to previous reports [62 (link),63 (link),64 (link),65 (link)].
Exionlc ad
Manufactured by Thermo Fisher Scientific
Sourced in China
The ExionLC AD is a high-performance liquid chromatography (HPLC) system designed for analytical applications. It provides accurate and reliable separation of complex sample mixtures. The system features advanced automation capabilities and can be integrated with various detection technologies to support a wide range of analytical workflows.
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Lab products found in correlation
6500 triple quadrupole, by Thermo Fisher Scientific (12 mentions)
4500 q trap, by Thermo Fisher Scientific (2 mentions)
Alkaline phosphatase, by Takara Bio (2 mentions)
Phosphodiesterase 1, by Merck Group (2 mentions)
Acquity uplc hss t3 c18, by Waters Corporation (2 mentions)
Qtrap 6500, by AB Sciex (2 mentions)
Scaa 104, by ANPEL (1 mentions)
Cnwbond carbon gcb spe cartridge, by ANPEL (1 mentions)
Analyst 1, by AB Sciex (1 mentions)
Sb c18 reversed phase column, by Agilent Technologies (1 mentions)
Acquity uplc hss t3 c18 1.8 μm, by Waters Corporation (1 mentions)
Chloroform, by Merck Group (1 mentions)
S1 nuclease, by Takara Bio (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Sw28ti rotor, by Beckman Coulter (1 mentions)
Quercetin 3 o glucoside, by Yuanye Bio-Technology (1 mentions)
Sunfire c18 column, by Waters Corporation (1 mentions)
Agilent 1290 hplc system, by Agilent Technologies (1 mentions)
Kaempferol 3 o glucoside, by Yuanye Bio-Technology (1 mentions)
6500 q trap, by Thermo Fisher Scientific (1 mentions)
18 protocols using exionlc ad
1
Quantification of Phytohormones in Plant Stem
2
Targeted Metabolite Profiling of Fruit Skin
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Metabolite profiling was conducted via a widely targeted metabolite method from Wuhan Metware Biotechnology Co., Ltd., (http://www.metware.cn/ , accessed on 21 December 2021), Wuhan, China. Freeze-dried fruit skin samples were powdered and analyzed via UPLC-ESI-MS/MS (UPLC, ExionLC™ AD’ https://sciex.com.cn/ , accessed on 21 December 2021; MS, Applied Biosystems 4500 Q TRAP, https://sciex.com.cn/ , accessed on 21 December 2021), Shanghai, China, identified by comparing the m/z values, the retention time (RT), and the fragmentation patterns with the standards in a self-compiled database [37 (link)]. Significantly changed metabolites (SCMs) were filtered according to Variable Importance in Projection (VIP) ≥ 1 and | Log2 (Fold Change)|≥ 1 [38 (link)]. Data underwent log transformation (log2) and mean centering before orthogonal PLS-DA analysis (OPLS-DA). Identified metabolites were annotated with the Kyoto Encyclopedia of Genes and Genomes (KEGG) Compound database and mapped to KEGG Pathways (http://www.kegg.jp/kegg/pathway.html , accessed on 21 December 2021) [39 (link)]. Metabolite sets enrichment analysis (MSEA) determined the pathway significance using the p-values of hypergeometric tests.
3
Metabolite Profiling of Frozen Plant Leaves
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The frozen leaves were grounded to powder and 100 mg powder was extracted overnight at 4°C with 0.6 ml 70% aqueous methanol, centrifuged at 10,000 g for 10 min, absorbed in CNWBOND Carbon-GCB SPE Cartridge (250 mg, 3 ml; ANPEL, Shanghai, China) and filtrated with a 0.22 μm SCAA-104 (ANPEL, Shanghai, China). The obtained extracts were then subjected to metabolites analysis with an ultra-performance liquid chromatography (UPLC)-ESI-MS/MS system (UPLC, ExionLC AD; MS, Applied Biosystems 6,500 Triple Quadrupole). The parameters were set as previously described (Zhu et al., 2017 (link)), and a mixture of supernatant from each biological sample was used as a quality control (QC) sample to evaluate the stability of the system. The data was scaled and subjected to principal component analysis (PCA). Metabolites with variable important in projection (VIP) greater than or equal to 1 and absolute Log2FC (fold change) greater than or equal to 1 were deemed as significantly regulated and annotated against the KEGG database. K-means clustering was carried out with R software.
4
Lipidomic Profiling of Cellular Lipids
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The lipidomic profiling was performed using an Ultra-Performance Liquid Chromatography Mass Spectrometry system (UPLC, ExionLC AD; MS, Applied Biosystems SCIEX 6500+QTRAP; Wuhan Metware Biotechnology Co., Ltd, China). Briefly, cells were harvested and lipids were extracted using a methyl tertbutyl ether and methanol mixture. The supernatants were loaded into the LC-MS/MS system. Analyst 1.6.3 software (AB Sciex) was applied to analysis the mass spectrometric data. Built-in Metware database (MWDB) and the public database of metabolite information were performed for the qualitative analysis. The lipid metabolite structural analysis was mainly based on the MassBank, KNAPSAcK, HMDB, LIPID MAPS and METLIN databases. The first screening was focused on the significant features, with a p value < 0.05, and fold change >1.2. The web-based tools BioPAN in LIPID MAPS (https://lipidmaps.org/biopan/ ) and MetaboAnalyst 5.0 were applied for the enrichment analysis. Fatty acid and phosphatidylinositol concentrations were detected using a Nonesterified Free fatty acids assay kit (A042-2-1, Nanjing Jiancheng Bioengineering institute) and Human Phosphatidylinositol (PI) ELISA Kit (TL-E1418H, Telenbiotech), respectively, according to the manufacturer’s instructions.
5
UPLC-MS/MS Analysis of Unknown Compounds
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Sample extracts were analyzed using a UPLC-ESI-MS /MS system (UPLC, ExionLC™AD' https://sciex.com.cn/ ; MS, Applied Biosystems 4500 Q TRAP, https://sciex.com.cn/ )29 (link). The UPLC conditions are described as follows: (1) Column: Agilent SB-C18 reversed-phase column with a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 µm was used, (2) Mobile phase A: methanol–water (90:10, containing 0.1% formic acid and 0.1% acetic acid), pH adjusted to 3.5, (3) Mobile phase B: pure methanol, (4) Gradient elution conditions: start with 10% mobile phase B; 0–0.5 min, linear gradient from 10 to 40% mobile phase B; 0.5–3 min, mobile phase B maintained at 40%; 3–3.5 min, linear gradient from 40 to 90% mobile phase B; 3.5–4 min, mobile phase B maintained at 90%; 4–4.5 min, linear gradient from 90% back to 10% mobile phase B; 4.5–6 min, mobile phase B maintained at 10%, (5) Flow rate: 0.3 mL/min, (6) Injection volume: 3 µL, and (7) Temperature: room temperature. The efflux was alternatively applied to an ESI-triple quadrupole-linear ion trap (QTRAP)- MS.
6
RNA Nucleoside Quantification by LC-MS/MS
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Briefly, adding buffer S1 nuclease (Takara Biotechnology, Dalian, Liaoning, China), Alkaline Phosphatase (Takara Biotechnology) and Phosphodiesterase I (Sigma‐Aldrich) into 1 μg RNA, then the mixture was incubated at 37°C. After the RNA was digested into nucleosides completely, the mixture was extracted with chloroform (Sigma‐Aldrich). The resulting aqueous layer was collected for analysis with LC‐MS/MS. The sample extracts were analyzed using an UltraPerformanceLiquid (UPLC)‐MS/MS system (UPLC, ExionLC™ AD, Applied Biosystems 6500 Triple Quadrupole; oster City, CA, USA). The analytical conditions were as follows, LC: column, Waters ACQUITY UPLC HSS T3 C18 (1.8 μm; Waters, Milford, MA, USA); solvent system, water (2 mmol/L NH4HCO3): methanol (2 mmol/L NH4HCO3); gradient program, 95:5V/V at 0 min, 95:5V/V at 1 min, 5:95 V/V at 9 min, 5:95 V/V at 11 min, 95:5 V/V at 11.1 min, 95:5 V/V at 14 min; flow rate, 0.30 mL/min; temperature, 40°C; injection volume: 10 μL. The effluent was alternatively connected to an Electrospray Ionization‐triple quadrupole‐linear ion trap. A specific set of multiple reaction monitoring transitions were monitored for each period according to the metabolites eluted within this period.
7
Nucleoside Extraction and Liquid Chromatography-Tandem Mass Spectrometry
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The nucleoside mixture after the RNA guy was extracted with chloroform. The resulting aqueous layer was collected and used for liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) (UPLC, ExionLC AD; MS, Applied Biosystems 6500 Triple Quadruole) analysis.
8
Endogenous Cytokinin Analysis in Plants
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Plant samples were the same as described in 2.6. The measurement of endogenous cytokinins was performed as described with minor modifications (Dobrev and Vankova, 2012 (link)). In brief, 50 mg of each fresh plant material was frozen in liquid nitrogen, ground into powder, and extracted with methanol/water/formic acid (15:4:1, V/V/V). The combined extracts were evaporated to dryness under nitrogen gas stream, reconstituted in 80% methanol (V/V), and filtered (PTFE, 0.22 μm; Anpel). The extracts were analyzed using an UPLC-ESI-MS/MS system (UPLC, ExionLC™ AD; MS, Applied Biosystems 6500 Triple). The experiments were relegated to Wuhan Metware Biotechnology Co., Ltd. (Wuhan). Three replicates of each assay were performed under the same conditions.
9
Quantifying Phytohormones in Seed Tissues
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The contents of IAA, cytokinin and ABA were measured with the seed tissues of DAP20 from ssm1 and LH11. Approximately 1.0 g of each sample was rapidly frozen in liquid nitrogen and ground into a powder, then extracted with 1 mL methanol/water/formic acid (15:4:1, V/V/V). The quantification of endogenous phytohormones were conducted according to the manufacturer’s instructions (Wuhan Metware Biotechnology Co., Ltd., Wuhan, China) and using the external standard method. The sample extracts were analyzed using an LC-ESI-MS/MS system (UHPLC, ExionLC™ AD; MS, Applied Biosystems 6500 Triple Quadrupole). The analytical conditions were as follows, HPLC: column, Waters ACQUITY UPLC HSS T3 C18 (100 mm × 2.1 mm i.d., 1.8 µm); solvent system, water with 0.04% acetic acid (A), acetonitrile with 0.04% acetic acid (B); gradient program, started at 5% B (0–1 min), increased to 95% B (1–8 min), 95% B (8–9 min), finaly ramped back to 5% B (9.1–12 min); flow rate, 0.35 mL/min; temperature, 40 °C; injection volume: 2 μL. Three biological replications were performed.
10
Quantitative Serum Metabolite Analysis
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Mouse serum (50 μL) was added with 200 μL methanol/acetonitrile. Ten microlitres of an internal standard mixed solution (1 μg/mL) were added to the extract as an internal standard for quantification. Samples were taken at − 20 °C for 10 min. After centrifuging for 10 min (12,000 r/min, 4 °C), the supernatant was evaporated to dryness and reconstituted in 100 μL of 50% methanol for further LC–MS/MS analysis. The analysis was performed using an LC–ESI–MS/MS system (UHPLC, ExionLC™ AD; MS, Applied Biosystems 6500 Triple Quadrupole).
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