Unsaturated phospholipid fatty acid chain position identification method

Abstract

The invention relates to the field of structural analysis, and mainly relates to an unsaturated phospholipid fatty acid chain position identification method. The invention relates to an unsaturated phospholipid fatty acid chain position identification method. The method comprises the following steps: analyzing a one-dimensional nuclear magnetic carbon spectrum of to-be-analyzed unsaturated phospholipid. The identification method provided by the invention is simple and rapid, does not damage the sample, does not need complex pretreatment on the sample, does not need derivatization, and can be used for direct detection. According to the technical scheme provided by the invention, the position information of the unsaturated phospholipid fatty acid chain can be obtained only by adopting a one-dimensional nuclear magnetic carbon spectrum under the condition that a standard substance is not used. The technical method provided by the invention can be easily applied to identification of the unsaturated phospholipid fatty acid chain position isomer.

Description

Technical Field

[0001] This application relates to the field of structural analysis, and mainly to a method for identifying the position of unsaturated phospholipid fatty acid chains. Background Technology

[0002] Phospholipids are essential components of cell membranes in organisms, playing crucial biological roles in energy storage, signal transduction, and metabolism. Phospholipid structural information exists at multiple levels, including lipid class, fatty acid chain length, the C=C position and stereochemistry (cis / trans configuration) of the fatty acid acyl group, and the stereospecific number (sn) position of the fatty acid chain, referred to as fatty acid chain position. Lipid function is closely related to its structure, and many studies have shown a correlation between changes in lipid composition and structure in vivo and metabolism and disease. For example, the length and unsaturation of fatty acid chains significantly affect the fluidity of biological membranes, revealing the importance of lipid structural characterization for studying their role in biological metabolism and disease diagnosis. However, in-depth structural analysis of phospholipids still presents challenges, such as the accurate identification of phospholipid fatty acid chain positions. Studies have observed significant differences in fatty acid chain position isomers across different tissues, with one fatty acid chain position isomer being enriched in the presynaptic membrane, suggesting its unique physiological function. Therefore, in-depth structural analysis of phospholipids is of great significance.

[0003] Mass spectrometry-based analytical methods have been applied to lipid structure analysis. Currently, for the identification of the fatty acid chain positions of unsaturated phospholipids, existing mass spectrometry-based techniques mainly fall into two categories. One category employs novel dissociation methods, such as ozone-induced dissociation (OzID), ultraviolet photodissociation (UVPD), and electron collision excitation (EEIO) of organic ions, which can identify the positions of unsaturated phospholipid fatty acid chains. The drawback of this type of technique is that it requires special modifications to the mass spectrometer to achieve the novel dissociation function, making it difficult to widely apply to ordinary mass spectrometers. The other type of mass spectrometry-based technique employs a strategy of derivatizing unsaturated phospholipids, such as using the Paternò-Büchi reaction. The derivatized lipids can be identified by collision-induced dissociation (CID) in ordinary mass spectrometers. The drawback of this type of technique is that it requires derivatization of the analyte phospholipid, and the derivatization efficiency is relatively low, failing to convert all phospholipids into a single derivatized product.

[0004] In addition, enzymatic hydrolysis is another method for analyzing the position of phospholipid fatty acid chains. This method mainly relies on specific hydrolysis or alcoholysis using lipases (such as phospholipase A2). By analyzing the lipids after enzymatic hydrolysis, the position of unsaturated phospholipid fatty acid chains can be identified. However, enzymatic hydrolysis requires a long reaction time and involves complex analytical procedures.

[0005] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this application is to provide a method for identifying the position of unsaturated phospholipid fatty acid chains. The aim is to provide a new position identification method that does not require complex sample pretreatment, does not require derivatization, can be directly detected, and is non-destructive to the sample.

[0007] The technical solution of this application is as follows: A method for identifying the position of fatty acid chains in unsaturated phospholipids includes the following steps: analyzing the one-dimensional carbon NMR spectrum of the unsaturated phospholipid to be analyzed.

[0008] Furthermore, a method for identifying the position of unsaturated phospholipid fatty acid chains includes the following steps: Obtain the one-dimensional carbon NMR spectrum of the unsaturated phospholipid to be analyzed; The chemical shift of the carbonyl carbon in the low-field region was obtained; Determine the chemical shift distribution of the carbonyl carbon in the low-field region; This enables the identification of the fatty acid chain positions in the unsaturated phospholipids to be analyzed.

[0009] Furthermore, after obtaining the one-dimensional carbon NMR spectrum of the unsaturated phospholipid to be analyzed, the chemical shift of the carbonyl carbon in the low-field region of the one-dimensional carbon NMR spectrum of the unsaturated phospholipid to be analyzed is set according to the peak region of the carbonyl carbon in the fatty acid chain.

[0010] Furthermore, the chemical shift distribution of the carbonyl carbon in the fatty acid chain of the unsaturated phospholipid to be analyzed is divided into two distribution patterns: "high outside and low inside" and "high inside and low outside".

[0011] Furthermore, the positions of the fatty acid chains to be identified in the unsaturated phospholipids to be analyzed are divided into... sn 1 and sn 2.

[0012] Furthermore, the unsaturated phospholipid to be analyzed is dissolved in a deuterated reagent, and then a one-dimensional carbon NMR spectrum of the unsaturated phospholipid to be analyzed is obtained.

[0013] Furthermore, the deuterated reagent includes at least deuterated chloroform.

[0014] The identification method provided in this application is simple, rapid, and non-destructive to the sample. It requires no complex pretreatment or derivatization and can be directly detected. Furthermore, the technical solution provided in this application can obtain the positional information of unsaturated phospholipid fatty acid chains using only one-dimensional carbon NMR spectroscopy without the use of standards. The technical method proposed in this application can be easily applied to the identification of positional isomers of unsaturated phospholipid fatty acid chains.

[0015] Compared with the prior art, this application has the following beneficial effects: 1. This application is simple and fast, and does not require cumbersome pretreatment of the unsaturated phospholipids to be analyzed, such as derivatization, and can be directly used for NMR experiments.

[0016] 2. This application can obtain the position information of unsaturated phospholipid fatty acid chains by using one-dimensional carbon NMR spectroscopy without the use of standards. Attached Figure Description

[0017] Figure 1 This is a technical roadmap for a method for identifying the position of unsaturated phospholipid fatty acid chains according to this application.

[0018] Figure 2 The chemical structure diagram and chemical shift peak diagram of the unsaturated phospholipid to be analyzed in Example 1 are shown.

[0019] Figure 3 The chemical structure diagram and chemical shift peak diagram of the unsaturated phospholipid to be analyzed in Example 2 are shown. Detailed Implementation

[0020] This application provides a method for identifying the position of unsaturated phospholipid fatty acid chains. To make the purpose, technical solution, and effects of this application clearer and more explicit, the following provides a more detailed description. It should be understood that the specific embodiments described herein are merely illustrative of this application and are not intended to limit this application.

[0021] Reference Figure 1 This is a technical roadmap for a method for identifying the position of unsaturated phospholipid fatty acid chains provided in this application.

[0022] This application provides a method for identifying the position of unsaturated phospholipid fatty acid chains, comprising the following steps: The unsaturated phospholipid to be analyzed is dissolved in a deuterated reagent, and then a one-dimensional carbon NMR spectrum of the analyte is obtained, i.e., the analyte's... 13 C spectrum.

[0023] The chemical shifts of carbonyl carbons in the low-field region were obtained from one-dimensional carbon NMR spectra.

[0024] The chemical shift distribution of the carbonyl carbon in the fatty acid chain of the unsaturated phospholipids to be analyzed is divided into two types: "high on the outside and low on the inside" and "high on the inside and low on the outside".

[0025] The positions of the fatty acid chains to be identified in the unsaturated phospholipids to be analyzed are divided into... sn 1 and sn 2.

[0026] Determine the chemical shift distribution of carbonyl carbons in the low-field region to identify the positions of fatty acid chains in the unsaturated phospholipids to be analyzed.

[0027] In this application, the deuterated reagent is deuterated chloroform, but is not limited to deuterated chloroform.

[0028] The identification method provided in this application does not require derivatization of the unsaturated phospholipids to be analyzed and can be directly performed by NMR experiments.

[0029] The present application will be further described below through specific embodiments.

[0030] Example 1 The unsaturated phospholipid selected in this embodiment is PC 16:0 / 18:1 (9Z). The chemical structure of this unsaturated phospholipid is as follows: Figure 2 As shown in (a), the C18:1 position of the unsaturated fatty acid chain is... sn 2. The position of C16:0 in the saturated fatty acid chain is... sn 1.

[0031] The nuclear magnetic resonance spectrometer used in this embodiment is the Bruker Avance Neo 600 nuclear magnetic resonance spectrometer, and the deuterated reagent used is deuterated chloroform.

[0032] The unsaturated phospholipid to be analyzed was dissolved in deuterated chloroform (the ratio is not particularly important), transferred to a 5 mm NMR tube, and then subjected to NMR analysis directly. An NMR experiment was set up to obtain the one-dimensional carbon spectrum of the unsaturated phospholipid to be analyzed. 13 (C spectrum). The region of interest in the carbon spectrum is the area where the carbonyl carbon ions exhibit peaks in the low-field region. For example... Figure 2 As shown in (b), the chemical shift peaks of the corresponding carbonyl carbons in the two fatty acid chains of this unsaturated phospholipid can be obtained from the low-field region of the carbon spectrum at PC 16:0 / 18:1 (9Z). Observation Figure 2 (b) The chemical shift characteristics of the carbonyl carbon were used to determine the chemical shift distribution of the carbonyl carbon in the unsaturated phospholipid fatty acid chain. Figure 2 (a) It can be known that when the C18:1 position of the unsaturated phospholipid fatty acid chain is... sn At position 2, the distance ratio between the corresponding carbonyl carbon and the phosphate group at the head of the phospholipid is C18:1. sn 1 is closer. Similarly, when the C16:0 position of the unsaturated phospholipid fatty acid chain is... sn At position 1, the distance between the corresponding carbonyl carbon and the phosphate group at the head of the phospholipid is in the ratio of C16:0. sn 2. Further away. In compounds, highly electronegative groups affect the electron cloud density of neighboring carbon atoms, causing changes in the chemical shift of the carbon atoms. Phosphate groups have strong electronegativity and an electron-withdrawing effect. This effect leads to a decrease in the electron cloud density of neighboring carbon atoms, causing the chemical shift of neighboring carbon atoms to shift to a lower field, resulting in an increase in the chemical shift value.

[0033] The unsaturated phospholipids selected in this embodiment sn If the fatty acid chain attached to position 2 is C18:1, then the distance between the carbonyl carbon and the phosphate group in the C18:1 chain will be greater than that in the C18:1 chain. sn When the carbonyl carbon is closer to the position 1, the corresponding carbonyl carbon's chemical position shifts further to a lower field, resulting in a larger chemical shift value.

[0034] The unsaturated phospholipids selected in this embodiment sn If the fatty acid chain attached to position 1 is C16:0, then the distance between the carbonyl carbon and the phosphate group in C16:0 will be greater than that in C16:0. sn The position is further away, which causes the chemical site of the corresponding carbonyl carbon to shift further to a higher field, resulting in a smaller chemical shift value.

[0035] Since the unsaturated phospholipid selected in this embodiment is difficult to obtain as 100% pure PC 16:0 / 18:1(9Z), in other words, it will be mixed with a small amount of the corresponding isomer, namely PC 18:1(9Z) / 16:0. Therefore, the chemical shift distribution of the carbonyl carbon in the fatty acid chain of this unsaturated phospholipid can be obtained as "high on the outside and low on the inside", that is, the two outer peaks are high and the two inner peaks are low, such as... Figure 2 As shown in (b). The chemical shift of the carbonyl carbon in the unsaturated phospholipid fatty acid chain was determined to be a "high on the outside, low on the inside" distribution, thus allowing identification of the position of the C18:1 fatty acid chain in the unsaturated phospholipid. sn 2. The position of the C16:0 fatty acid chain is... sn 1.

[0036] Example 2 The sample used in this embodiment is an unsaturated phospholipid with a PC 18:1(9Z) / 16:0 ratio. The chemical structure of this unsaturated phospholipid is as follows: Figure 3 As shown in (a), the C18:1 position of the unsaturated fatty acid chain is... sn 1. The position of C16:0 in the saturated fatty acid chain is... sn 2.

[0037] The nuclear magnetic resonance spectrometer used in this embodiment is also the Bruker Avance Neo 600 nuclear magnetic resonance spectrometer, and the deuterated reagent used is deuterated chloroform.

[0038] The unsaturated phospholipids to be analyzed were dissolved in deuterated chloroform and transferred to a 5 mm NMR tube for direct NMR analysis. A one-dimensional carbon spectrum of the unsaturated phospholipids to be analyzed was obtained using an NMR experiment. 13 (C spectrum). The region of interest in the carbon spectrum is the area where the carbonyl carbon ions exhibit peaks in the low-field region. For example... Figure 3As shown in (b), the chemical shift peaks of the corresponding carbonyl carbons in the two fatty acid chains of this unsaturated phospholipid can be obtained from the low-field region of the carbon spectrum at PC 18:1(9Z) / 16:0. Observation Figure 3 (b) The chemical shift characteristics of the carbonyl carbon were used to determine the chemical shift distribution of the carbonyl carbon in the unsaturated phospholipid fatty acid chain. Figure 3 (a) It can be known that when the C18:1 position of the unsaturated phospholipid fatty acid chain is... sn At position 1, the distance ratio between the carbonyl carbon and the phosphate group at the head of the phospholipid is 1 (C18:1). sn 2 is further away. Similarly, when the position of C16:0 in the unsaturated phospholipid fatty acid chain is... sn At position 2, the distance between the corresponding carbonyl carbon and the phosphate group at the head of the phospholipid is in the C16:0 ratio. sn 1. Closer. In compounds, highly electronegative groups affect the electron cloud density of neighboring carbon atoms, causing a change in the chemical shift value of the carbon atom. Phosphate groups have strong electronegativity and an electron-withdrawing effect. This effect leads to a decrease in the electron cloud density of neighboring carbon atoms, causing the chemical shift of neighboring carbon atoms to shift to a lower field, resulting in an increase in the chemical shift value.

[0039] The unsaturated phospholipids selected in this embodiment sn If the fatty acid chain attached to position 2 is C16:0, then the distance between the carbonyl carbon and the phosphate group in C16:0 will be greater than that in C16:0. sn When the carbonyl carbon is closer to the position 1, the corresponding carbonyl carbon's chemical position shifts further to a lower field, resulting in a larger chemical shift value.

[0040] The unsaturated phospholipids selected in this embodiment sn If the fatty acid chain attached to position 1 is C18:1, then the distance between the carbonyl carbon and the phosphate group in the C18:1 chain will be greater than that in the C18:1 chain. sn The position is further away, which causes the chemical site of the corresponding carbonyl carbon to shift further to a higher field, resulting in a smaller chemical shift value.

[0041] Since the unsaturated phospholipid selected in this embodiment is not 100% pure PC 18:1(9Z) / 16:0, it contains a small amount of the corresponding isomer, PC 16:0 / 18:1(9Z). Therefore, the chemical shift distribution of the carbonyl carbon in the fatty acid chain of this unsaturated phospholipid can be obtained as "high inside and low outside", that is, the two inner peaks are high and the two outer peaks are low, as shown in the example. Figure 3 As shown in (b). The chemical shift of the carbonyl carbon in the unsaturated phospholipid fatty acid chain was determined to be a "high inside, low outside" distribution, thus allowing identification of the position of the C18:1 fatty acid chain in the unsaturated phospholipid. sn 1. The position of the C16:0 fatty acid chain is... sn 2.

[0042] It should be understood that the application of this application is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of this application.

Claims

1. A method for identifying the position of unsaturated phospholipid fatty acid chains, characterized in that, Includes the following steps: The one-dimensional carbon NMR spectrum of the unsaturated phospholipids to be analyzed was performed.

2. The method for identifying the position of unsaturated phospholipid fatty acid chains according to claim 1, characterized in that, Includes the following steps: Obtain the one-dimensional carbon NMR spectrum of the unsaturated phospholipid to be analyzed; The chemical shift of the carbonyl carbon in the low-field region was obtained; Determine the chemical shift distribution of the carbonyl carbon in the low-field region; This enables the identification of the fatty acid chain positions in the unsaturated phospholipids to be analyzed.

3. The method for identifying the position of unsaturated phospholipid fatty acid chains according to claim 1 or 2, characterized in that, After obtaining the one-dimensional carbon NMR spectrum of the unsaturated phospholipid to be analyzed, the chemical shift of the carbonyl carbon in the low field region of the one-dimensional carbon NMR spectrum of the unsaturated phospholipid to be analyzed is set according to the peak region of the carbonyl carbon in the fatty acid chain.

4. The method for identifying the position of unsaturated phospholipid fatty acid chains according to claim 2, characterized in that, The chemical shift distribution of the carbonyl carbon in the unsaturated phospholipid fatty acid chain to be analyzed is divided into two types: "high on the outside and low on the inside" and "high on the inside and low on the outside".

5. The method for identifying the position of unsaturated phospholipid fatty acid chains according to claim 1, characterized in that, The positions of the fatty acid chains to be identified in the unsaturated phospholipids to be analyzed are divided into... sn 1 and sn 2.

6. The method for identifying the position of unsaturated phospholipid fatty acid chains according to claim 1, characterized in that, The unsaturated phospholipid to be analyzed was dissolved in a deuterated reagent, and then a one-dimensional carbon NMR spectrum of the unsaturated phospholipid to be analyzed was obtained.

7. The method for identifying the position of unsaturated phospholipid fatty acid chains according to claim 6, characterized in that, The deuterated reagent includes at least deuterated chloroform.

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