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Pretreatment method and quantitative detection method for endogenous sugar phosphate compound in plant sample

A phosphate sugar, endogenous technology, applied in the field of analytical chemistry, can solve the problems of low sensitivity, affecting the accuracy of quantification, damage to the mass spectrometer, etc., and achieve the effect of improving the signal response of the mass spectrometer, ensuring the accuracy, and improving the detection sensitivity.

Active Publication Date: 2019-02-19
WUHAN UNIV
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  • Application Information

AI Technical Summary

Problems solved by technology

However, high concentrations of salt are often added to the chromatographic mobile phase of strong cation exchange chromatography, which reduces the detection sensitivity of mass spectrometry to a certain extent, and also causes damage to the mass spectrometer to a certain extent.
Hydrophilic interaction chromatography can solve this problem very well. Phosphate sugar compounds have a strong retention on the hydrophilic column. At the same time, the mobile phase used in the hydrophilic column can also perfectly match the mass spectrometer. However, using a hydrophilic column to separate the phosphate sugar There is still the problem of not being able to base-separate the isomers, which will affect the accuracy of quantification
[0005] Second, due to the poor ionization efficiency of T6P, the fragmentation behavior on CID is not good, resulting in poor signal response on MS, and the complex plant matrix will interfere with subsequent detection, resulting in low sensitivity

Method used

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  • Pretreatment method and quantitative detection method for endogenous sugar phosphate compound in plant sample
  • Pretreatment method and quantitative detection method for endogenous sugar phosphate compound in plant sample
  • Pretreatment method and quantitative detection method for endogenous sugar phosphate compound in plant sample

Examples

Experimental program
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Effect test

Embodiment 1

[0040] In this example, four standard substances were used as analytes to verify their chromatographic separation. Prepare mixed standards of glucose-1-phosphate, glucose-6-phosphate, sucrose-6-phosphate, and trehalose-6-phosphate, each standard 0.2ng dissolved in 300 μL of 50 mM borate buffered saline (pH 6.8); Add 180 μL of 8-DMQ DMSO solution (0.014 mM) to the resulting solution, then place it at room temperature to shake for 40 minutes, centrifuge (10000×g) for 5 minutes to take the supernatant; then take 2 μL of the supernatant to pass through the HILIC column for mass spectrometry analysis .

[0041] As shown in Figure 1, the two pairs of isomers are well separated, have good chromatographic retention behavior, and the chromatographic peak shape is good.

Embodiment 2

[0043] In this example, four standard substances were used as analytes to verify the improvement of sensitivity before and after derivatization.

[0044] First, prepare mixed standards of glucose-1-phosphate, glucose-6-phosphate, sucrose-6-phosphate, and trehalose-6-phosphate, and dissolve 0.2 ng of each standard in 300 μL of 50 mM borate buffered saline solution (pH 6.8 ); add 180μL of 8-DMQ DMSO solution (0.014mM) to the resulting solution, then place it at room temperature to shake for 40min, centrifuge (10000×g) for 5min to get the supernatant; then take 2μL of the supernatant to pass through the HILIC column Enter mass spectrometry.

[0045] In addition, prepare mixed standards of glucose-1-phosphate, glucose-6-phosphate, sucrose-6-phosphate, and trehalose-6-phosphate, and dissolve 0.2 ng of each standard in 480 μL of 50 mM borate buffered saline (pH 6.8 ); after that, 2 μL of the resulting solution was passed through a HILIC chromatographic column for mass spectrometry ...

Embodiment 3

[0048] A pretreatment method for endogenous phosphate sugar in a plant sample, mainly comprising the following steps:

[0049] 1) Accurately weigh 1mg of rice leaves, 5mg of rice roots, and 10mg of Arabidopsis leaves and grind them into powder with liquid nitrogen as plant samples;

[0050] Add the isotope internal standard [ 13 C 6 ] Phosphate-6-glucose 25ng and 0.25mL iced chloroform / acetonitrile 3:7 (v / v) were mixed evenly; after vortex mixing, placed in a refrigerator at -20°C for extraction for 2 hours, and vortexed occasionally; after that, Add 0.2mL of ice water and vortex for 3min. After centrifugation (10000×g) for 5min, take out the upper aqueous phase and put it in another centrifuge tube; add 0.2mL of ice water again to repeat the above steps, and combine the two extracts to obtain the sample extraction solution;

[0051] 2) Blow dry the sample extraction solution obtained above under nitrogen, then reconstitute with 200 μL of 50 mM boric acid buffered saline so...

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Abstract

The invention discloses an analysis method for a variety of sugar phosphate compounds including endogenous trehalose-6-phosphate in a plant sample. The method comprises the following steps of: extracting the endogenous sugar phosphates through a solvent; adsorbing and removing the hydrophobic impurities in the extracted solution through a reversed-phase SPE column; using the diazo groups in a derivatization reagent to carry out derivatization reaction on the phosphate groups common to the sugar phosphate compounds; and in combination with the liquid chromatography and mass spectrometry, realizing the separation detection of the endogenous sugar phosphate isomers in the plant sample. The analysis method is simple, accurate and high in throughput, can achieve the baseline separation of the sugar phosphate isomers on a hydrophilic chromatographic column and guarantee the higher detection sensitivity, and finally can realize the separation detection of the endogenous sugar phosphates in amicroplant tissue sample.

Description

technical field [0001] The invention relates to a method for pretreatment and quantitative analysis of endogenous sugar phosphate compounds in plant samples, belonging to the field of analytical chemistry. Background technique [0002] Phosphosaccharides are important signaling molecules in plants and play important roles in many physiological processes including seed development, juvenile-adult transition, floral transition, bud branching, senescence and embryonic maturation. However, the mechanism of action of phosphate sugars in plants is still unknown. Therefore, the establishment of accurate and highly sensitive analytical methods is the key to exploring the role of phosphate sugar compounds in plants. [0003] So far, many analytical methods have been developed for the analysis and detection of phosphate sugar compounds in plants. Among these methods, the LC-MS method has become the mainstream method for the analysis of phosphate sugars due to its high sensitivity an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/14G01N30/72
CPCG01N30/02G01N30/06G01N30/14G01N30/72G01N2030/067
Inventor 冯钰锜罗晓彤蔡保东
Owner WUHAN UNIV
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