In order to analysis the trend for change in phytohormones, leaf samples of d0 (mixed samples of CK-D0 and LT-D0) and d15 (CK-D15 and LT-D15) with three biological replicates were collected for phytohormone quantification. Approximately 500 mg of each sample was rapidly frozen in liquid nitrogen. The extraction and quantification of endogenous ACC (ethylene precursors) and JA were performed using an LC-ESI-MS/MS system (UPLC, Shim-pack UFLC SHIMADZU CBM30A system, http://www.shimadzu.com.cn/ , access date 12 October 2021, Kyoto, Japan; MS/MS, Applied Biosystems, Foster City, CA, 6500 Quadrupole Trap, http://www.appliedbiosystems.com.cn/ , access date 12 October 2021) by Wuhan Metware Biotechnology Co., Ltd. (Wuhan, China) [115 (link),116 (link),117 (link),118 (link),119 (link)].
6500 quadrupole trap
Manufactured by Thermo Fisher Scientific
Sourced in Japan
The 6500 Quadrupole Trap is a high-performance mass spectrometer instrument designed for advanced analytical applications. It utilizes quadrupole ion trap technology to capture and analyze ions, providing accurate mass measurements and detailed structural information. The 6500 Quadrupole Trap is capable of performing a range of mass spectrometry techniques, including full-scan, tandem mass spectrometry (MS/MS), and multiple reaction monitoring (MRM) analyses.
Automatically generated - may contain errors
5 protocols using 6500 quadrupole trap
1
Quantification of Phytohormone Dynamics
2
LC-ESI-MS/MS Analysis of Plant Hormones
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The extracts were analyzed using an LC-ESI-MS/MS system (Ultra Performance Liquid Chromatography, UPLC, Shim-pack UFLC SHIMADZU CBM30A system; Tandem mass spectrometry, MS/MS, Applied Biosystems 6500 Quadrupole Trap). Hormone separation was performed with a HPLC Waters ACQUITY UPLC HSS T3 C18 column (1.8 µm, 2.1 mm × 100 mm) using UP water with 0.04% acetic acid (solvent A) and acetonitrile with 0.04% acetic acid (solvent B) as mobile phases. The following parameters were used for HPLC: flow rate of 0.35 mL/min; column temperature 40°C; 5 μL injection; method: 0 min (95% A: 5% B), 0–11 min linear gradient to 5% A:95% B, 11–12 min (5% A: 95% B), 12–12.1 min (95% A: 5% B), and 12.1–15 min (95% A:5% B). The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (Q TRAP)-MS. The ESI source operation parameters were as follows: ion source, turbo spray; source temperature, 500°C; ion spray voltage (IS), 5500 V; curtain gas, 35.0 psi; and collision gas medium. DP and CE for individual MRM transitions were performed with further DP and CE optimization. A specific set of MRM transitions was monitored for each period according to the plant hormones eluted during this period.
3
Bioactive Compound Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Take an appropriate amount of sample in a centrifuge tube, and add 80% methanolic water containing 1% formic acid, vortex, and sonicate. Centrifuge (4 °C, 15,000 r/min, 5 min) and take the supernatant over the filter membrane containing H5 purification powder on the machine for detection. The data acquisition system included high-performance liquid chromatography (HPLC, SHIMADZU-20A) and tandem mass spectrometry (MS/MS, applied biosystems 6500 quadrupole trap). The mass spectrometry data were processed using the software Analyst, and the content was calculated using the standard curve.
4
Extraction and Quantification of Plant Hormones
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The samples were ground in liquid nitrogen to dry powder. The 1.5 g of powder was put in a glass test tube and added isopropanol-water-hydrochloric acid mixed extract into it, followed by shaking at low temperature for 30 min. Then, dichloromethane was added, followed by shaking at low temperature for 30 min and centrifugation at 13000 r/min for 5 min at low temperature. The lower organic phase was removed, protected from light, and dried with nitrogen. The organic phase was reconstituted with methanol (0.1% formic acid); centrifuged at 4°C for 10 min (13000 r/min), and the supernatant was taken to 0.22 µm filter membrane. For HPLC-MS/MS detection, the extraction process was performed on an ice box at 4°C throughout the operation. Methanol (0.1% formic acid) was used to prepare different gradients of IAA, ABA, Zeatin, and GA3 standard solutions for the solvent, and the standard curve in practice linear outliers was excluded from the equation. The data acquisition system mainly included high-performance liquid chromatography (High-Performance Liquid Chromatography, HPLC) (Agilent 1290, https://www.agilent.com/ ) and tandem mass spectrometry (Tandem mass spectrometry, MS/MS) (Applied Biosystems 6500 Quadrupole Trap, https://sciex.com.cn/ ). Mass spectral data were processed using the software Analyst.
5
UPLC-MS/MS Analysis of Hormones
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Extracts obtained from above were subjected to an Ultra-High-Performance Liquid Chromatography (UPLC) (Shim-pack UFLC SHIMADZU CBM3OA, http//www.shimadzu.com.cn) and a Tandem Mass Spectrometry (Ms/ms) (Applied Biosystems 6500 Quadrupole Trap, http://www.applied biosystems.com.cn/). The detailed procedures described in (Ma et al. 2008 (link)) were followed.
The liquid phase conditions were:
(1) Chromatographic column: Waters ACQUITY UPLC HSS T3 C18 1.8 um, 2.1 m × 100 mm;
(2) Mobile phase: water phase was ultrapure water (0.04% formic acid added) in the water phase, acetonitrile (0.04% formic acid added) in the organic phase;
(3) Elution gradient: 0 min water/acetonitrile (90:10V/V), 5.0 min 40:60V/V, 70 min 40:60V/V, 7.01 min 90:10V/V and 10.0 min 90:10V/V;
(4) The ow rate of 0.35 mL/min, column temperature of 40℃, and injection volume of 2 micron.
Mass spectrometry conditions were as followed: electrospray ionization (ESI) temperature was 500℃, mass spectrometry voltage was 5500V, curtain gas was 35 psi, the collision-activated dissociation parameter was set to medium in the dissociation, and each ion pair was scanned according to the optimized cluster voltage and collision energy. The hormonal content obtained in the analysis was expressed as mg/g fresh weight.
The liquid phase conditions were:
(1) Chromatographic column: Waters ACQUITY UPLC HSS T3 C18 1.8 um, 2.1 m × 100 mm;
(2) Mobile phase: water phase was ultrapure water (0.04% formic acid added) in the water phase, acetonitrile (0.04% formic acid added) in the organic phase;
(3) Elution gradient: 0 min water/acetonitrile (90:10V/V), 5.0 min 40:60V/V, 70 min 40:60V/V, 7.01 min 90:10V/V and 10.0 min 90:10V/V;
(4) The ow rate of 0.35 mL/min, column temperature of 40℃, and injection volume of 2 micron.
Mass spectrometry conditions were as followed: electrospray ionization (ESI) temperature was 500℃, mass spectrometry voltage was 5500V, curtain gas was 35 psi, the collision-activated dissociation parameter was set to medium in the dissociation, and each ion pair was scanned according to the optimized cluster voltage and collision energy. The hormonal content obtained in the analysis was expressed as mg/g fresh weight.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!