Pretreatment method for enrichment and solid-phase derivatization of sugar phosphate substances

A technology for phosphate sugars and sugar substances, which is applied in the direction of measuring devices, instruments, and material analysis through electromagnetic means, so as to improve the detection sensitivity of mass spectrometry, shorten the time required, and avoid the use of excessive derivative reagents.

Active Publication Date: 2017-10-24
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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So far, there have been no reports on the pretreatment method of enrichment and solid-phase der

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  • Pretreatment method for enrichment and solid-phase derivatization of sugar phosphate substances
  • Pretreatment method for enrichment and solid-phase derivatization of sugar phosphate substances
  • Pretreatment method for enrichment and solid-phase derivatization of sugar phosphate substances

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Embodiment 1

[0016] Embodiment 1. immobilized metal affinity chromatography material SiO 2 @PD-Ti 4 The synthesis of + and its enrichment and derivation process for phosphate sugars.

[0017] The first step to synthesize immobilized metal affinity chromatography material SiO 2 @PD-Ti 4+ : 200mg SiO2 2 (particle size 3μm) was dispersed in 40mL of dopamine aqueous solution with a concentration of 2mg / mL, and 40mL Tris buffer (pH 8.5) was quickly added under magnetic stirring, and reacted for 10 hours at a water bath temperature of 40°C under magnetic stirring conditions, and SiO was obtained after washing with water. 2 @PD microspheres. Then SiO 2 @PD dispersed in 80mL of Ti(SO 4 ) 2 In the solution, the reaction was continued for 3 hours at a water bath temperature of 40°C under magnetic stirring conditions, and SiO was obtained after washing with water. 2 @PD-Ti 4+ Microspheres. Synthesis schematic see figure 1 .

[0018] The second enrichment derivation process: take 6-phospha...

Embodiment 2

[0019] Example 2. Selective Enrichment of Phosphosaccharides in Derived Cells and LC-MS Analysis

[0020](1) Cell pretreatment: 7721 liver cancer cells were taken out from the incubator, quenched instantly with liquid nitrogen, and washed 3 times with PBS buffer. Add 1mL of methanol, scrape the cells and transfer them to the EP tube, vortex for 30s, then add 400μL of ultrapure water, vortex for 30s, finally add 1mL of chloroform, and vortex for 30s. After standing still for 1 min, centrifuge at 15000 rpm / min for 10 min. Take 900 μL of supernatant and freeze-dry. Add 4mL sample solution (by ACN / H 2 O (65 / 35, v / v) solution was prepared by adding a final concentration of 2% (v / v) formic acid (FA) and a final mass fraction of 0.4% glutamic acid) for reconstitution.

[0021] (2) Material pretreatment process: take 10mg SiO 2 @PD-Ti 4+ Material, with sample solution (by ACN / H 2 O (65 / 35, v / v) solution was prepared by adding a final concentration of 2% (v / v) formic acid (FA) an...

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Abstract

The invention belongs to the technical field of inorganic materials and analysis and particularly relates to a pretreatment method for enrichment and solid-phase derivatization of sugar phosphate substances by a metal ion affinity chromatography material (SiO2@PD-Ti<4+>). The pretreatment method comprises the following steps: firstly, enriching the sugar phosphate substances on SiO2@PD-Ti<4+> microspheres by a metal chelating action; secondly, carrying out solid-phase derivatization on enriched sugar phosphate substances. According to the method, retention and separation degree of the sugar phosphate substances on reversed phase liquid chromatography can be improved, and the mass spectrometric detection sensitivity of the sugar phosphate substances with low abundance can be improved. The pretreatment method disclosed by the invention has the advantages of simple and quick pretreatment process, good repeatability, low detection limit and the like.

Description

technical field [0001] The invention belongs to the technical field of inorganic materials and analysis, and is a kind of material based on metal ion affinity chromatography (SiO 2 @PD-Ti 4+ ) pretreatment method for enrichment and solid-phase derivatization of phosphate sugars and its application. Background technique [0002] Phosphoric sugars such as glucose-6-phosphate, ribose-5-phosphate, erythrose-4-phosphate and glyceraldehyde-3-phosphate play important roles in many life processes. They are important intermediate metabolites in central carbon metabolism, such as glycolysis, gluconeogenesis, pentose phosphate pathway and tricarboxylic acid cycle metabolic pathway, and can participate in the regulation of tumor energy metabolism. At present, liquid chromatography-mass spectrometry (LC-MS) technology has been widely used in the detection of phosphate sugars due to its advantages of high sensitivity and high resolution. However, due to the strong polarity of phosphate...

Claims

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Application Information

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IPC IPC(8): G01N30/06G01N30/89G01N30/02G01N27/62
CPCG01N27/626G01N30/02G01N30/06G01N30/89G01N2030/027G01N2030/067
Inventor 许国旺秦倩石先哲王博弘
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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