Lipid molecular structure analysis method based on extreme ultraviolet laser dissociation and mass spectrometric detection

Abstract

The invention relates to a novel analysis method for carrying out lipid gas phase dissociation by adopting pulse extreme ultraviolet laser and characterizing a lipid molecular structure by analyzing dissociated fragment ions through mass spectrometry. A lipid sample enters a gas phase through electrospray ionization, then lipid molecules with accurately selected mass are efficiently excited and dissociated by adopting extreme ultraviolet laser to generate fragment ions with sequence and structural characteristics, and finally the fragment ions are detected through mass spectrum. According to the method, efficient extreme ultraviolet light dissociation of lipid molecules can be achieved, structural characteristics such as head groups, sn positions, double bond positions and cis-trans isomerism of the lipid molecules can be accurately determined through fragment ion spectrogram analysis, and accurate identification and distinguishing of complex lipid isomers are achieved.

Description

Technical Field

[0001] The present invention relates to a method for efficiently dissociating lipids by extreme ultraviolet laser for mass spectrometry. Background Art

[0002] In biology, lipids have many important functions and can be broadly classified into categories such as energy storage (e.g., triacylglycerols in adipose tissue), structural components (e.g., glycerophospholipids in membrane bilayers), and signaling molecules (e.g., diacylglycerol and ceramide). Therefore, lipids play a regulatory role in biological processes such as maintaining homeostasis, metabolism, signal transduction, and apoptosis. Lipids can not only provide insights into changes in metabolic processes caused by environmental stimuli but also serve as biomarkers for identifying disease states. Alterations in lipid metabolism play a key role in the pathogenesis of many diseases, including atherosclerosis, Alzheimer's disease, and cancer. Generally, individual lipids exert their biochemical effects through their molecular structures. For example, phospholipids with different head groups are specifically distributed within cells and play important roles in lipid-sterol interactions that affect the structure of membrane bilayers; the degree of unsaturation and chain branching of acyl chains in lipids can have a certain impact on the fluidity of membranes. However, one of the most challenging tasks in lipidomics analysis is to characterize isomeric lipid species at the level of "structurally defined molecular lipids", including the assignment of stereospecific numbering (sn)-regional chemistry and enantiomeric configuration of acyl / alkyl chains, as well as determining the precise position and stereochemistry (i.e., cis / trans) of double bonds in their unsaturated acyl / alkyl chains. To comprehensively understand complex biological networks, the identification of fine structures at the lipid molecular level is essential.

[0003] Mass spectrometry is one of the core scientific instruments in the field of lipidomics analysis. Electrospray ionization mass spectrometry (ESI-MS) combined with tandem mass spectrometry (MS / MS) for structural analysis has become a key platform and technology in lipidomics. Most traditional MS / MS-based structure identification methods use low-energy collision-induced dissociation (CID) or high-energy collision-induced dissociation (HCD), and the dissociation of lipid ions is achieved through multiple collisions between the electric field traction and gas molecules. This dissociation mode is a slow excitation mode of internal energy accumulation, and the chemical bond with the lowest bond energy in the ion usually breaks preferentially, which can only complete the identification at the fatty acyl level and it is difficult to obtain information such as the sn position, double bond position, and cis-trans isomerism. Based on the interaction between photons and lipids, photodissociation is a newly emerging mass spectrometry dissociation mode. The photoexcitation and dissociation of lipid ions are related to the wavelength and pulse width of the light used. Infrared lasers require multi-photon excitation to dissociate lipid ions due to their low photon energy. Ultraviolet lasers can achieve single-photon dissociation processes due to their high photon energy. The currently most developed 193-nm ultraviolet laser dissociation technology can generate fragment ions related to double bonds in lipid dissociation, but it is still difficult to obtain sn information. Therefore, further reducing the wavelength of the excitation light source and increasing the energy of single photons are effective methods to achieve efficient lipid dissociation, and it is expected to achieve precise analysis of information such as lipid head groups, sn positions, double bond positions, and cis-trans isomerism. Summary of the Invention

[0004] The present invention relates to a lipid dissociation method based on extreme ultraviolet laser. By introducing 70-160 nm extreme ultraviolet laser generated by a free electron laser into mass spectrometry, efficient mass spectrometry dissociation of lipids can be achieved, generating characteristic fragment ions, and precise determination of information such as lipid head groups, sn positions, double bond positions, and cis-trans isomerism can be realized through the fragment ion spectra.

[0005] The present invention provides, but is not limited to, the following new methods for characterizing the structure of lipid molecules based on pulsed extreme ultraviolet laser dissociation and mass spectrometry detection:

[0006] 1) Sample preparation method: Take appropriate amounts of lipid standards and dissolve them in chloroform / methanol solution (v / v, 2 / 1) to obtain a stock solution with a concentration of 1 mg / mL and store it in a -20 °C refrigerator. Before the experiment, pipette an appropriate amount of the target lipid stock solution into pure methanol to obtain an electrospray-compatible lipid methanol solution with a concentration of 10 μM.

[0007] 2) Mass spectrometry detection conditions: The lipid sample is ionized in a microfluidic electrospray mode using an infusion pump and then introduced into mass spectrometry for analysis. The mass spectrometry operates in the positive ion mode. Spray voltage: 3800 volts; source temperature: 275 °C; resolution: 240000; RFLens: 30%; AGC: 500;

[0008] Maximum injection time: 100 milliseconds.

[0009] 3) Laser conditions: The wavelength range of the pulsed laser is from 70 nm to 160 nm, the pulse width is from 10 femtoseconds to 100 nanoseconds, the pulse repetition frequency is from 1 Hz to 100,000 Hz, and the pulse intensity is from 0.1 microjoule to 1000 microjoules.

[0010] 4) Data acquisition: The mass-to-charge ratios of lipid ions are confirmed by full-scan mass spectrometry to obtain the mass-to-charge ratios of the hydrogenated peak, sodium-added peak, and potassium-added peak. Subsequently, the linear ion trap is used to selectively enrich each lipid adduct ion. The enriched lipid ions are injected into the extreme ultraviolet laser dissociation trap composed of multiple rods, and they are efficiently dissociated with extreme ultraviolet lasers of different wavelengths. Finally, the electrostatic field orbitrap mass analyzer (Orbitrap) is used to perform high-resolution detection on the generated fragment ions and collect the corresponding data.

[0011] The present invention can achieve efficient extreme ultraviolet light dissociation of lipid molecules. Through the analysis of the fragment ion spectra, the structural characteristics such as the head group, sn position, double bond position, and cis-trans isomerism of lipid molecules can be accurately determined, realizing the accurate identification and differentiation of complex lipid isomers.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] Based on the action of short-pulse high-energy photon beams generated by extreme ultraviolet lasers on lipid ions, the present invention first proves that extreme ultraviolet lasers can achieve efficient mass spectrometry dissociation of lipids, and its dissociation characteristics are completely different from other known mass spectrometry dissociation modes. The short-pulse, high-brightness extreme ultraviolet laser can achieve sufficient dissociation of intact lipid ions in the gas phase without the need to modify the lipid structure before analysis. Its dissociation efficiency is far superior to other mass spectrometry dissociation modes, and it can simultaneously and accurately analyze the head group, sn position, double bond position, and cis-trans isomer information of lipids. Description of the Drawings

[0014] Figure 1 Extreme ultraviolet laser dissociation mass spectra of the hydrogenated peaks of PC(16:0,18:1) at 130 nm and 150 nm under 1 pulse and 5 pulses respectively

[0015] Figure 2 Mass spectra of the hydrogenated peak of PC(16:0,18:1): (A) 193 nm UVPD (1 pulse, 2 mJ, pulse width 5 ns, pulse repetition frequency 10 Hz) and (B) HCD (NCE 30)

[0016] Figure 3 Extreme ultraviolet laser dissociation mass spectrum of the sodium-added peak of DG(18:1) at 120 nm (1 pulse, 5 uJ);

[0017] Figure 4Mass spectra of DG(18:1) sodium adduct peak by (A) 193 nm UVPD (1 pulse, 2 mJ) and (B) HCD (NCE 30) Detailed implementation manners

[0018] The present invention will be described in detail below in conjunction with specific embodiments. The following examples will help those skilled in the art and researchers to further understand the present invention, but do not constitute any limitation to the present invention. Any modification made in any form within the scope of the claims of the present invention is still within the scope of protection of the claims of the present invention.

[0019] The basic components of the mass spectrometer used in the experiment include but are not limited to a microfluidic electrospray source, a quadrupole, a dual-pressure linear ion trap, an electrostatic field orbitrap mass analyzer (Orbitrap), etc. (along the ion flow direction). The pulsed extreme ultraviolet laser is introduced from the center along the axial direction of the dual-pressure linear ion trap.

[0020] Example 1

[0021] Dissociation of phosphatidylcholine PC(16:0,18:1) based on extreme ultraviolet laser

[0022] (1) Sample preparation

[0023] The PC(16:0,18:1) sample was dissolved in electrospray-compatible methanol at a concentration of 10 μM.

[0024] (2) Extreme ultraviolet laser dissociation mass spectrometry analysis

[0025] The lipid sample was ionized by an injection pump in a microfluidic electrospray mode and then introduced into the mass spectrometry analysis. The mass spectrometry worked in the positive ion mode. Spray voltage: 3800 volts; source temperature: 275 degrees Celsius; resolution: 240000; RF Lens: 30%; AGC: 500; maximum injection time: 100 milliseconds.

[0026] The mass-to-charge ratio of lipid ions (150 - 1000) was confirmed by a full scan of the mass spectrometry. The mass-to-charge ratio information of lipid molecules was obtained in the first-order spectrum of the mass spectrometry, and the mass-to-charge ratios of the hydrogen adduct peak, sodium adduct peak, and potassium adduct peak were obtained. Subsequently, each lipid adduct ion was selected and enriched by a quadrupole. The enriched lipid ions were injected into the extreme ultraviolet laser dissociation trap (dual-pressure linear ion trap), and were efficiently dissociated by 130 nm and 150 nm extreme ultraviolet lasers respectively. The working frequency of the extreme ultraviolet laser was 10 Hz, the single-pulse energy was 5 μJ, the pulse width was 10 femtoseconds, and the dissociation time was 1 millisecond. Finally, the electrostatic field orbitrap mass analyzer (Orbitrap) was used to perform high-resolution detection on the generated fragment ions, 200 spectra were accumulated, and 20 spectra corresponding to the collected data (including the mass-to-charge ratio of the ions and their relative intensities) were acquired.

[0027] At the same time, conventional higher-energy collisional dissociation (HCD) (the process and conditions are the same as the above process, the difference from the above process is that no 130 nm and 150 nm extreme ultraviolet lasers are introduced into the dissociation trap) and 193 nm ultraviolet photodissociation (the process and conditions are the same as the above process, the difference from the above process is that a 193 nm extreme ultraviolet laser is used to replace the 130 nm and 150 nm extreme ultraviolet lasers) are used as a comparison to characterize that the extreme ultraviolet laser dissociation proposed by the present invention is a brand-new mass spectrometry dissociation method, which is significantly different from the existing mass spectrometry dissociation methods.

[0028] (3) Data analysis

[0029] Based on the PC(16:0,18:1) molecular formula, the theoretical molecular mass and theoretical mass-to-charge ratio of the fragment ions are calculated. The collected data is opened with the Thermo Fisher Scientific FreeStyle software (FreeStyle 1.7, Thermo Fisher) to obtain the molecular weight and mass-to-charge ratio information of the fragment ions generated by extreme ultraviolet laser dissociation. With a tolerance error of 20 ppm, the detected molecular weight is compared with the theoretical molecular weight to confirm the composition of the generated fragment ions and obtain the characterization information of PC, enabling accurate determination of information such as lipid head groups, sn positions, double bond positions, and cis-trans isomers. From Figure 1 and 2 it can be seen that both 193 nm ultraviolet laser and conventional higher-energy collisional dissociation can generate head group information ions, and the 193 nm ultraviolet laser can generate fragment ions related to double bonds compared with HCD, but it is still difficult to obtain sn information. Compared with 193 nm ultraviolet photodissociation and HCD, the 130 nm extreme ultraviolet laser dissociation has an increased number of ion fragment species, including not only head group information ions, but also the 491.34 fragment revealing the sn position information and the 632.43 and 634.44 fragments directly fragmented from the carbon chain double bond. Through the obtained characteristic fragment ions, the structure of unknown PC lipids can be identified and analyzed.

[0030] Example 2

[0031] Extreme ultraviolet laser dissociation of diacylglycerol DG(18:1)

[0032] (1) Sample preparation

[0033] DG(18:1) is dissolved in electrospray-compatible methanol at a concentration of 10 μM.

[0034] (2) Extreme ultraviolet laser dissociation mass spectrometry analysis

[0035] The DG(18:1) sample was ionized in the microfluidic electrospray mode using an injection pump and then introduced into mass spectrometry analysis. The mass spectrometry was operated in the positive ion mode. Spray voltage: 3800 volts; source temperature: 275 degrees Celsius; resolution: 240000; RF Lens: 30%; AGC: 500; maximum injection time: 100 milliseconds. The mass spectrometry full scan confirmed the mass-to-charge ratios (150 - 1000) of different adduct forms of this ion, and then the quadrupole was used to select and enrich the ions. The enriched ions were injected into a linear ion trap and efficiently dissociated using a 120-nanometer extreme ultraviolet laser. The working frequency of the extreme ultraviolet laser was 10 Hz, the single pulse energy was 5 μJ, the pulse width was 100 ns, and the dissociation time was 1 ms. Finally, the generated fragment ions were detected with high resolution using an electrostatic field Orbitrap mass analyzer, 200 spectra were accumulated, and 20 spectra corresponding to the collected data (including the mass-to-charge ratio of the ions and their relative intensities) were acquired.

[0036] At the same time, conventional collision-induced dissociation (HCD) and 193-nanometer ultraviolet photodissociation were used as comparisons to characterize that the extreme ultraviolet laser dissociation proposed in the present invention is a brand-new mass spectrometry dissociation method, which is significantly different from the existing mass spectrometry dissociation methods.

[0037] (3) Data analysis

[0038] Based on the DG(18:1) molecular formula, the theoretical molecular mass and theoretical mass-to-charge ratio of the fragment ions were calculated. The collected data was opened using Thermo Fisher's FreeStyle software (FreeStyle 1.7, Thermo Fisher) to obtain the molecular weight and mass-to-charge ratio information of the fragment ions generated by extreme ultraviolet laser dissociation. The detected molecular weight was compared with the theoretical molecular weight with a tolerance error of 20 ppm to confirm the composition of the generated fragment ions and obtain the characterization information of DG(18:1). From Figure 3 and 4 it can be seen that, similar to Example 1, compared with 193-nanometer ultraviolet photodissociation and HCD, the number of ion fragment types increased during 120-nanometer extreme ultraviolet laser dissociation. On this basis, high-intensity 347.25 fragments of glycerol backbone fragmentation and 515.37 and 517.38 fragments of direct fragmentation of carbon chain double bonds, which could not be obtained by 193-nanometer ultraviolet photodissociation and HCD, were also obtained. Through such information, the structure of glycerides can be clearly obtained.

Claims

1. A method for analyzing the structure of lipid molecules based on extreme ultraviolet laser dissociation and mass spectrometry detection, characterized in that: The lipid sample enters the gas phase through electrospray, and the mass-to-charge ratio information of lipid molecules is obtained in the first-order spectrum of the mass spectrometer. The hydrogenated peak, sodium-added peak, and potassium-added peak are precisely mass-selected and focused in the ion trap. The focused lipid molecules are excited and induced to dissociate by introducing pulsed extreme ultraviolet laser in the ion trap to generate sequence- and structure-specific fragment ions. Finally, the fragment ions are detected and the spectra are recorded by the mass spectrometer to characterize the lipid molecular structure.

2. The method according to claim 1, characterized in that: The wavelength range of the pulsed laser is from 70 nm to 160 nm, the pulse width is from 10 femtoseconds to 100 nanoseconds, the pulse repetition frequency is from 1 Hz to 100,000 Hz, and the pulse intensity is from 0.1 microjoule to 1000 microjoules.

3. The method according to claim 1, characterized in that: The lipid sample enters the gas phase through electrospray and generates various types of sequence- and structure-specific fragment ions, including radical fragment ions, such as head groups and fatty chains, under the excitation of a single pulse or multiple pulses of the pulsed extreme ultraviolet laser.

4. The method according to claim 1, characterized in that: The intensity of the photoionization peak of lipid molecules under the action of the laser or the sum intensity of the dissociation fragment ions is selective for different wavelengths, obtaining extreme ultraviolet photoionization spectra and extreme ultraviolet photodissociation spectra; The precise characterization of lipid structures, including multi-level structures such as the positions of head groups and double bonds and the differentiation of fatty chains, can be achieved through the analysis of extreme ultraviolet laser dissociation fragments of lipid molecules, realizing the precise qualitative differentiation of complex lipid isomers.

5. The method according to any one of claims 1-4, characterized in that: Lipid molecules include, but are not limited to, phosphatidylcholine (PC) and / or diacylglycerol (DG).

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