Method for detecting bis-phospholipid in high flux manner

A dual phospholipid, high-throughput technology, applied in the field of analytical chemistry, can solve the problems of difficult separation of chromatography, separation of secondary fragment information, lack of fatty acid chain information of secondary fragments, etc., to improve detection sensitivity and simplify pretreatment steps Effect

Active Publication Date: 2019-03-01
TSINGHUA UNIV
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  • Abstract
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Problems solved by technology

[0003] The structural formulas of BMP and PG are as follows. They are isomers of each other, and it is difficult to distinguish them in chromatography in the absence of standard products. The secondary fragments in positive ion mode lack fatty acid chain information and cannot be further characterized. Although negative ion mode has Fatty acid chain information, but the secondary fragment information is consistent and it is difficult to distinguish them
The hydrophobic end of BMP tends to connect to polyunsaturated fatty acids, while PG tends to connect to saturated or monounsaturated fatty acids, but the combination of fatty acid chains and double bond numbers in biological samples makes the detection of BMP more complicated
However, so far there has been no report on high-throughput BMP analysis based on liquid Chromatograph Mass Spectrometry (LC-MS)

Method used

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  • Method for detecting bis-phospholipid in high flux manner
  • Method for detecting bis-phospholipid in high flux manner
  • Method for detecting bis-phospholipid in high flux manner

Examples

Experimental program
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Effect test

Embodiment 1

[0052] Establishment of a high-throughput analytical method based on ultra-high performance liquid chromatography-obitrap mass spectrometer

[0053] The process of establishing a high-throughput analytical method based on ultra-high performance liquid chromatography-obitrap mass spectrometer is as follows: figure 2 , the specific implementation steps are as follows:

[0054] The standard products BMP and PG and the corresponding internal standards were purchased from avanti company. The standard is firstly dissolved in a mixture of chloroform and methanol, and then stored in a low temperature environment (-20°C to 80°C). When it is necessary to perform actual sample inspection or preparation of a standard curve, it is then diluted to the required concentration with a mixture of methylene chloride and methanol. The concentration is the working solution.

[0055] 1. Sample preparation and pretreatment:

[0056] Preparation of BMP standard and PG standard: Take 100 μl of BMP ...

Embodiment 2

[0070] Example 2: Methodological investigation of high-throughput bisphosphate detection based on UHPLC / Obitrap

[0071] The tissue samples were mixed into quality control (Quality Control, QC) samples, which were pretreated according to the following different investigation objects.

[0072] Repeatability inspection of analytical methods

[0073] Take 50mg of fresh tissue samples, add 500ul chloroform:methanol:water mixture (20:10:5, v:v:v) to extract lipids, take the organic phase and transfer it to a new sample tube to dry, and use derivatization reagent IV Methylsilane was derivatized and then spin-dried. The spin-dried samples were redissolved in 100 μl of chloroform-methanol mixture for LC-MS analysis. Repeat processing of 8 QC tissue samples was done in parallel.

[0074] Eight QC samples were subjected to high-throughput analysis of BMP on the UHPLC / Obitrap platform. Peak matching was performed on the measured data using Tracfinder software, and 9 BMP lipid molecul...

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Abstract

The invention discloses a method for detecting bis-phospholipid in a high flux manner. The method comprises the following steps: separately derivatizating a bis-phospholipid standard product and a phosphatidyl glycerol standard product, separating and detecting the bis-phospholipid and phosphatidyl glycerol standard product by using liquid chromatography tandem mass spectrometry, and establishinga high-flux liquid chromatography tandem mass spectrometry database related to the bis-phospholipid; derivatizating a sample, separating and detecting the sample by using the liquid chromatography tandem mass spectrometry, so as to obtain bis-phospholipid characteristic ions of the sample; and searching the obtained bis-phospholipid characteristic ions in the high-flux liquid chromatography tandemmass spectrometry database, and acquiring the information related to the bis-phospholipid in the sample. According to the method, by establishing the high-resolution database, the high-flux analysisfor BMP can be realized; and by adopting the derivatization method, not only can the signal intensity of the BMP be improved, but also the interference of isomeride can be reduced, and the accuracy ofa data analysis result and the repetition of the data analysis result can be improved.

Description

technical field [0001] This application relates to the field of analytical chemistry, especially but not limited to a method for high-throughput detection of bisphospholipids based on ultra-high performance liquid chromatography / high-resolution mass spectrometry. Background technique [0002] Bis (monoacylglycero) phosphate (BMP) and phosphatidylglycerol (PG) are structural isomers, but they have different biological functions in vivo. PG is a class of acidic phospholipids and is an important component of cell membranes; BMP is a class of markers that target the inner membranes of late endosomes and lysosomes and are often used to detect these organelles. The unique structure of BMP makes it resistant to the degradation of most phospholipases. The research results show that the increase of BMP is closely related to many diseases. Therefore, more and more people pay attention to the relationship between BMP and diseases. The metabolic profile analysis of BMP substances has ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/72
CPCG01N30/02G01N30/06G01N30/72G01N2030/067
Inventor 刘晓蕙许丽娜王雪颖焦玉佩
Owner TSINGHUA UNIV
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