KR20190035048A - Qualitative and quantitative analysis method of in vivo lipid using isotope substitution and methylation method - Google Patents

Abstract

The present invention provides a qualitative and quantitative analysis method of lipid in a body which comprises: an extraction step of extracting lipid in a body; an alkylation step of introducing an isotope-substituted alkyl group to the extracted lipid; and an analysis step of analyzing the alkylated lipid using a liquid chromatography-electrospray ionization-mass spectrometer system.

Description

TECHNICAL FIELD The present invention relates to an in vivo lipid-based isotope substitution and methylation method,

The present invention relates to a qualitative and quantitative analysis method of lipids in vivo, and more particularly, to a method of qualitative and quantitative analysis of lipids in vivo using isotope substitution and methylation methods.

Lipids have traditionally been known to play a role in biomembrane constituents and energy storage. However, lipid has attracted much attention because it has been reported as a biomarker related to diseases in recent years as well as various signal transduction among cells. The research on lipidomics, which means the study of analyzing these lipids, is actively being conducted.

For example, in the case of type 2 diabetes, there is insulin resistance. As a cause thereof, excessive lipid accumulation occurs in non-adipose tissue, and lipotoxicity which is generated is pointed out. Therefore, . Many studies have shown that glycerol lipids such as diacylglycerol and triacylglycerol show a great change in diabetic patients, but phosphatidic acid (PA) and cardiolipin, which are known to be involved in cell death and mitochondrial function, To analyze the presence of trace amounts of phospholipids, including phospholipids, remains a challenge.

An object of the present invention is to provide an analytical method capable of qualitatively and quantitatively analyzing a phospholipid and a cardiolipin present in a trace amount in a biological sample.

In order to achieve the above-mentioned object, the present invention provides an extracting step of extracting lipids in vivo; An alkylation step of introducing an iso-substituted alkyl group into the extracted lipid; And analyzing the alkylated lipids using a liquid chromatography-electrospray ionization-mass spectrometer system.

In the present invention, the isotope may be deuterium.

In the present invention, the alkylation may be methylation.

In the present invention, the lipid may be a phospholipid.

In the present invention, the methylation of the phosphate group of the lipid can be achieved.

In the extraction step and alkylation step of the present invention may use one or more of MeOD, DCl and D ₂ O.

In the analysis step of the present invention, a mobile phase containing an ionization mediator may be used.

In the present invention, the ionization medium may be at least one selected from ammonium formate, ammonium hydroxide, and ammonium acetate.

In the present invention, the liquid chromatography-electrospray ionization-mass spectrometer system comprises a pump; Analytical column; valve; Metal wire; A microcross connected to the pump, the analysis column, the valve and the metal wire, respectively; And a pressure capillary connected to the valve.

In the analysis step of the present invention, the pressure capillary can be adjusted during the elution of the lipids, allowing the mobile phase to flow in the direction of the analytical column and the mass spectrometer at a rate of 300 to 500 nL / min.

The present invention develops a phospholipid quantitative analysis method using an isotope substitution-methyl group introduction method and a liquid chromatography-electrospray ionization-mass spectrometer, thereby effectively qualitatively and quantitatively analyzing phospholipid and cardiolipin present in a biological sample in a trace amount can do.

1 shows isotopic substitution-methylation, 1) shows the structure of phospholipids before and after methylation, and 2) shows a series of methylation processes using TMSD (trimethylsilyldiazomethane).
Figure 2 shows a liquid chromatography-electrospray ionization-mass spectrometer system.
FIG. 3 is a chromatogram showing 18 kinds of phospholipid standard substances in the case of methylation and in case of methylation, in which 1) is not methylation and 2) is methylation.
Figure 4 shows the relative ratios of hydrogen substitution-methylation and deuterated-substituted methylated phospholipids.
Figure 5 shows the qualitative analysis of methylated cardiolipin in the DU145 cell line.
6 shows the influence of the ionization medium on the moving image on the ionization efficiency.
Figure 7 shows the spectra of H- and D-methylated PL molecules obtained by nUPLC-ESI-MS ⁿ analysis.

Hereinafter, the present invention will be described in detail.

The lipid analysis method according to the present invention comprises: an extraction step of extracting lipids in vivo; An alkylation step of introducing an iso-substituted alkyl group into the extracted lipid; And an analytical step of analyzing the alkylated lipids using a liquid chromatography-electrospray ionization-mass spectrometry system.

Extraction step, methanol (MeOH), MTBE (Methyl Tertiary Butyl Ether), can extract the lipid in vivo using HCl or the like, wherein preferably at least one of MeOD, DCl and D ₂ O, more preferably both Can be used. The living body can be the cells and tissues of all living things including humans and animals. The lipid may preferably be a phospholipid.

In the alkylation step, an isotope-labeled alkyl group is introduced into the extracted lipid. The isotope may preferably be deuterium (D). The alkylation may preferably be methylation. In particular, the methylation of the phosphate group of the lipid can be achieved. In the alkylation step, methanol and (trimethylsilyl) diazomethane (TMSD) may be used, and at least one of MeOD and D ₂ O may be used.

In the analysis phase, alkylated lipids are analyzed using a liquid chromatography (LC) - electrospray ionization (ESI) - mass spectrometer (MS) system. The LC may be an ultra high performance LC (UPLC). The LC may utilize two or more mobile phases (e. G., Hydrophilic and hydrophobic mobile phases), wherein a concentration gradient of the mobile phase may be used. For the purpose of improving the ionization efficiency and the like, an ionization modifier may be added to the mobile phase. As the ionization medium, at least one selected from ammonium formate, ammonium hydroxide, and ammonium acetate can be used.

The LC-ESI-MS system includes an LC pump with an autosampler; An analytical column for lipid separation; Valves for flow control; Metal wire for electrospray ionization; A microcross connected to the pump, the analysis column, the valve and the metal wire, respectively; And a pressure capillary connected to the valve. For the purpose of improving the ionization efficiency and the like, the pressure capillary can be controlled during the elution of the lipids, and the mobile phase can be flowed at a rate of 300 to 500 nL / min toward the analyzing column and the mass spectrometer. The ionization efficiency of the lipids in the analysis step may be at least 92%, preferably 96%.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

Fig. 1-1 shows a structure of a phospholipid to be analyzed according to the present invention. The phospholipid is composed of two acyl chains and a phosphate group that are tailed to the glycerol basic skeleton and a head group (X) attached to the phosphate group. Which is a negative charge in oxygen. After methylation with TMSD and a deuterium-substituted reagent, a deuterated-methyl group (CHD ₂ ) can be introduced into the oxygen in the phosphate group. CH ₃ is introduced for general hydrogen-substituted methylation.

The methylation process described in FIGS. 1-2 is composed of two steps. First, the lipids are extracted, and then the reaction is performed with TMSD to perform methylation. The lipid in the biological sample can be extracted with an organic solvent consisting of methanol (MeOH) and MTBE and an aqueous solution HCl solution. Methanol and TMSD are added to the extracted lipids and reacted. After removing excess TMSD, methylated lipids can be finally obtained. To obtain deuterated methyl groups, MeOH and HCl solutions, which are deuterium-substituted reagents, may be used.

Figure 2 shows a liquid chromatography-electrospray ionization-mass spectrometer (LC-ESI-MS) system structure for analyzing methylated lipids. Ultra High Performance Liquid Chromatography (UPLC) can be carried out, for example, in mobile phase solvent A (90 ± 5% water, 10 ± 5% acetonitrile, v / v) and mobile phase solvent B (20 ± 5% methanol, 5%, isopropanol 60 ± 5%, v / v) can be used. The microcross connects the LC pump and the analytical column, a platinum wire is connected to connect the electrodes for electrospray ionization, and a valve can be connected to control the flow of the fluid. For example, the analytical column can be manufactured by filling a capillary having an inner diameter of 100 ± 10 μm with a C ₁₈ filler having a size of 1.7 ± 0.5 μm at a length of 7 ± 3 cm.

The extracted lipids can be injected into the column via an autosampler. First, in the valve connected state indicated by the solid line in FIG. 2, since the mobile phase 100% A from the pump moves all into the analysis column, the lipid can be distributed in the front part of the column. After that, the valve is changed to the dotted line state and the% of the mobile phase B is gradually increased, the lipids distributed to the column inlet are started to be eluted by the mobile phase B, and the order is changed according to the degree of interaction with the stationary phase (C ₁₈ ) And then enters the mass spectrometer. In this case, the solvent flowing out from the pump is in a unit of μL / min, but the pressure capillary is controlled to flow 300-500 nL / min toward the analyzing column and the mass analyzer, thereby minimizing the solvent in the mass spectrometry Ionization efficiency can be improved.

FIG. 3 shows the results of detection of a lipid standard material using the present invention. Dionex Ultimate 3000 RSLCnano system and LTQ Velose. In the case of no methylation, it is analyzed in the negative charge mode. Therefore, ammonium hydroxide medium (modifier) was added and analyzed. In the case of methylation, since it is analyzed in cationic mode, it was analyzed by adding ammonium formate medium. First, the valve position was adjusted so that the mobile phase A was moved only by the analytical column, and then the solution was flowed at 0.7 / / min for 10 minutes. After that, the valve position was changed to 60% mobile phase B for 0.1 minute, 75% mobile phase B for the next 5 minutes, 80% for 10 minutes at the rate of 7 μl / min, and finally to the 99% mobile phase B for 5 minutes Respectively. The fluid flow into the column was maintained at 300 nL / min.

The two chromatograms were tested by injecting 1 pmol of each lipid standard material, and the kinds of the reference materials were indicated by the respective peak numbers. It was confirmed that the retention time (retention time) increased when the methylation was performed. This indicates that the phospholipid structure was methylated, and the more nonpolar property became stronger, resulting in an increase in the interaction with the C ₁₈ particle during separation into LC do. The increase in overall peak intensity, including phosphatidic acid (PA) and cardiolipin (CL), may ultimately be attributed to increased ionization efficiency because PA or cardiolipin with increased nonpolarity is found in the droplet- It is expected that the ionization efficiency is increased due to the property of moving to the droplet-air interface. It can be predicted that the in vivo phospholipid present in the living body can be detected more effectively.

Figure 4 compares the general hydrogen substitution-methylation with the deuterated-methylated phospholipid. The nanoACQUITY UPLC system from Waters and the TSQ Vantage from Thermo Scientific were analyzed and analyzed by adding ammonium formate as the medium. First, the valve position was adjusted so that the mobile phase A was moved only by the analytical column, and then the solution was flowed at 0.7 / / min for 10 minutes. After that, the valve position was changed and analyzed by applying 70% mobile phase B for 0.1 minute at a rate of 14 / / min, 80% mobile phase B for the next 5 minutes and finally for 10 minutes with 100% mobile phase B. The fluid flow into the column was maintained at 300 nL / min. 4-1 is a representative example of the case of 16: 0/18: 1-PS. The hydrogen substitution-methylated sample and the deuterium-substituted methylated sample are mixed at a ratio of 2: 8, 4: 6, 5: 5, 6: 4, and 8: 2, respectively, on the y-axis. Through the good linearity, it can be seen that the general hydrogen substitution - methylation and deuterium - substitution - methylation are introduced into the phospholipid to the same degree, and the retention time of the two methylation is the same.

Figure 4-2 finally tests the matrix effect to apply this method to the prostate cancer cell line DU145. When the phospholipid standard material having an odd number chain is spiked to DU145 and analyzed as shown in FIG. 4-1, a value approximate to the expected ratio is obtained for phospholipids having various kinds of various acyl chains, It was confirmed that the normal hydrogen substitution-methylation and the deuterium substitution-methylation are introduced to the same extent in the actual biological sample.

After the methylation method using TMSD was applied to the DU145 cell line, qualitative analysis was carried out using a liquid chromatography-electrospray ionization-mass spectrometer. FIG. 5 shows 72: 5-cardiolipin as an example, and 72: 5-cardiolipin having an m / z value of 1501.5 in the mass spectrometry spectrum of FIG. 5-2 detected at 32.76 minutes in FIG. 5-1 . Four acyl chains in the cardiolipin can be identified using collision induced dissociation spectra (MS ⁿ spectra). First, each of the acyl chain pairs can be detected through the m / z values 601.5 and 603.5 shown in the MS ² spectrum in FIG. 5-3. In addition, the detailed structure in the MS ³ spectrum can be determined to obtain 72: 5-cardiolipin (18: 1, 18: 2) and (18: 1, 18: 1) - cardiolipin.

Finally, the qualitative analysis of the four phospholipids (PG, PS, PA, CL) in the DU145 cell line is shown in Table 1. When methylation was not detected, 79 were detected, but when methylation was detected, 112 were detected. In particular, CL could detect 25 more, which is far greater than the number detected in other literature, without methylation in the cell or tissue. Therefore, it can be concluded that cardiolipin present in a trace amount when analyzed by methylation can be analyzed. Table 1 shows the number of four phospholipids detected in DU145 cells.

Class Methylation X O PG 34 34 PS 15 15 PA 12 20 CL 18 43 Total 79 112

The present invention is based on a labeling method using an organic reagent. After labeling a large number of phospholipids present in a biological sample, it is separated through liquid chromatography equipped with a column made of a capillary filled with a filler having a hydrophobic property, And can be carried out in such a manner as to be detected by an electrospray ionization-mass spectrometer.

TMSD can be used as an organic reagent and methanol (MeOH), MTBE, HCl, water (H ₂ O), deuterated methanol (MeOD), DCl, deuterium substituted water (D ₂ O) May be required. Liquid chromatography can use two mobile solvents: a hydrophilic liquid, in which the phospholipid can be dispensed onto the stationary phase of the column, and the other a phospholipid is eluted in the column and then moved to an electrospray ionization- Lt; / RTI >

This quantitative analysis method has an advantage of detecting lipid species which was difficult to analyze due to an increase in sensitivity due to an increase in ionization efficiency of methylated phospholipids. In particular, it has an advantage that it can detect species present in trace amounts in cardiolipin, and can analyze the structure of four acyl chains having a complicated structure. In addition, the reproducibility problem due to ionization fluctuation can be reduced during the electrospray ionization-mass spectrometric analysis, thereby enabling more reliable quantitative analysis.

The existing phospholipid analysis method used an internal reference material as a method for correcting the ionization fluctuation in the electrospray ionization step before lipid molecules were injected into the mass spectrometer. Since the internal reference material is similar in nature to the lipid molecules in biological samples, but it is important to use materials that are not present in the sample, synthetic lipid molecules with an odd acyl chain are used as internal standards. However, the number of internal reference materials for calibrating numerous lipid molecules in biological samples is limited.

In order to supplement the lipid analysis method using the existing internal standard material, the isotope substitution-labeling method was used in the present invention, and a method of introducing a methyl group into the phospholipid was used. Further, since liquid chromatography is used in the present invention, it is possible to analyze a large number of lipid molecules in the living body. Combining isotope substitution-labeling with liquid chromatography can produce synergistic effects in in vivo lipid analysis. Without analyzing each of the two samples for comparative analysis, a single analysis can compare a large number of lipids and obtain more reproducible results.

In particular, cardiolipin has not been analyzed by labeling methods, and it has been difficult to analyze the lipid molecules, which have potential to serve as disease-related biomarkers as well as major constituents of the mitochondrial membrane. It can be seen that the present invention overcomes such a problem and has a great potential in the field of diagnostic medicine.

An important requirement for analytical methods used in the field of diagnostic medicine is that it is a reliable way to obtain reproducible results exactly for each test. In the present invention, since two samples to be compared and analyzed are detected by one analysis, errors occurring every analysis can be minimized. Also, the lipid extraction and methylation reagents used in the present invention are economically advantageous because they are cheaper than expensive internal standard materials.

Hereinafter, the present invention will be described more specifically by way of examples.

[Example]

In this example, lipid analysis using the isotope-labeled methylation (ILM) method was applied to nUPLC-ESI-MS ⁿ , and optimal settings including mobile phase and ionization media were established through various modifications. The method was evaluated by overall identification and quantification of intracellular PL using TMSD.

The systematic evaluation of the methylation efficiency of PL in the present example was carried out by the nUPLC-ESI-MS ⁿ by the SRM method using PL standards (phosphatidylglycerol (PG), lyso PG (LPG), phosphatidylinositol H-labeling using lyso PI (LPI), phosphatidylserine (PS), lysos PS (LPS), phosphatidic acid (PA), lyso PA (LPA), and cardiolipin (CL) (H / D) of methylated (-CH ₃ ) and D-labeled (-CHD ₂ ) methylation. We also investigated the effect of ionizing media on the detection of methylated PL in cationic mode. This method is a naturally occurring monosaccharide and has been shown to inhibit the proliferation of a variety of cancer cell lines by reducing the level of reactive oxygen species (ROS) in neutrophils after treatment of D-allose with hormone-refractory prostate cancer (HRPC) (DU145). ≪ / RTI > This example demonstrates that ILM in combination with nUPLC-ESI-MS ⁿ can serve as a powerful tool to quantify relative changes in the PL profile of D-allose treated prostate cancer cells. This example also focused on enhanced detection and rapid quantification of PS and CL closely related to apoptosis. ILM-based quantification of PL of cancer cells was compared to individual quantification using addition of internal reference material to analytical samples.

1. Experiment

(1) Materials and reagents

The mass spectrometer used was LTQ Velose and TSQ Vantage from Thermo Scientific (San Jose, CA, USA), Dionex Ultimate 3000 RSLCnano system from Thermo Scientific and nanoACQUITY UPLC from Waters (Milford, MA, USA). The analytical column and the connecting capillary were manufactured by Polymicro Technology, LLC (Phoenix, AZ, USA), and the outer diameter was 365 μm. The filler was 1.7 ㎛ BEH particles extracted from ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm) from Waters. All lipid standards used in the experiments were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). All HPLC grade solvents used in LC were purchased from Avantor Performance Materials (Center Vally, PA, USA) Ammonium formate and ammonium hydroxide modifier, mixed in a solvent, were purchased from Sigma-Aldrich (St. Louis, Mo., USA) to aid the process. CD ₃ OD (MeOD), D ₂ O, DCl and TMSD were purchased from Thermo Fisher Scientific (Waltham, Mass., USA) and HCl was purchased from Samchun Pure Chemicals Co. Ltd. (Seoul, Korea) and glacial acetic acid was purchased from Duksan Pure Chemicals Co. Ltd. (Ansan, Korea).

(2) Cell culture and lipid extraction

The DU145 HRPC cell line was obtained from Korean Cell Line Bank (Seoul, Korea). Cells are activated with 10% heat fire in the 100 mm petri dish fetal bovine serum (FBS) and 1% penicillin / streptomycin RPMI 1640 medium with hygromycin was added to and incubated with (Invitrogen, Carlsbad, CA), humidification, including 5% CO ₂ Lt; RTI ID = 0.0 > 37 C < / RTI > incubator for 48-72 hours. For D-allose treatment, 40 mM D-allose was added to the culture medium. When the cells occupied more than 90% of the dish area, cells were desorbed by the addition of 0.25% trypsin-EDTA. At this time, the cell concentration was about 6 × 10 ⁶ cells / mL.

Lipid extraction was performed according to the modified Folch method using MTBE / MEOH. In brief, the collected cell pellets were vacuum-dried and then MeOH (300 μl) was added. Samples were sonicated for 1 minute for homogenization. Then, 1 mL of MTBE was added to the homogenate and the sample was vortexed for 1 hour. Then, 250 [mu] l of 2.05 M HCl was added to the mixture. Samples were vortexed for 10 min and centrifuged at 1000 g for 10 min. The upper organic layer was transferred to a new tube and the remaining lower layer was re-extracted with 400 μl of the organic (upper) layer of a mixture of MTBE / MeOH / H ₂ O (10: 3: 2.5, v / v / v) Lt; RTI ID = 0.0 > 1000g < / RTI > for 5 minutes. The top layer was collected with the previously collected organic layer. After washing with vortexing aqueous (lower) layer on 500 ㎕ for 10 minutes and the combined organic layer the solvent mixture _{(MTBE / MeOH / H 2 O} ), and centrifuged for 5 minutes at 1000 g. The final organic layer was transferred to another tube for derivatization.

(3) Isotope-labeled methylation (ILM)

The methylation of the phosphate group of PL was initiated by addition of 50 [mu] l MEOH and 50 [mu] l TMSD (2 M in hexane, yellow) to the PL standard material mixture or the organic layer of the cell extract and then vortexed the mixture for 10 minutes at room temperature . To stop the reaction, 6 [mu] l of glacial acetic acid was added to the mixture, after which the yellow solution turned to a clear color. To this mixture, 500 μl of an aqueous (lower) layer of a fresh solvent mixture of MTBE / MEOH / H ₂ O (10: 3: 2.5, v / v / v) was added for washing. The mixture was vortexed for 10 min and centrifuged at 1000 g for 5 min. It was dissolved in: (1, v / v 9 ) - the final organic layer was vacuum dried and stored for MEOH / CHCl of 200 ㎕ _3. Was diluted with: (2, v / v 8 ) and the mixture MEOH / H ₂ O for nUPLC-ESI-MS / MS analysis. ILM was achieved by replacing MEOH, H ₂ O and HCl with CD ₃ OD, D ₂ O and DC ₁ , respectively, during all extraction and derivatization procedures.

(4) Analysis of nUPLC-ESI-MS ⁿ

Two nUPLC-ESI-MS / MS systems were used in this experiment. Non-target lipid identification was performed using a Dionex Ultimate 3000 RSLCnano system with an autosampler equipped with an LTQ Velos ion trap mass spectrometer from Thermo Scientific (San Jose, Calif.). SRM quantification of lipids was performed using Waters' nanoACQUITY UPLC system connected to Thermo Scientific's TSQ Vantage triple-stage quadrupole MS system. The analytical column was prepared in the laboratory. The mobile phase used for lipid separation is an A was: (6, v / v / v 2:: 2) MeOH / CH 3 CN / IPA, (9.5 0.5, v / v) H 2 O / CH 3 a CN and B 0.05% ammonium hydroxide (AH) as an ionization medium in anion mode and 5 mM ammonium formate (AF) in cation mode were added.

For non-target analysis of cellular lipids, 10 μg (2 μl, 5 μg / μl) of lipid extract was loaded onto the analytical column, at which time mobile phase A was flowed at 700 nL / min for 10 minutes and the switching valve was turned off . After the injection, the switching valve was turned on, the flow rate of the pump was set to 7 ㎕ / min, and the flow rate of the column was adjusted to 300 nL / min. Gradient elution I (running condition I) was carried out at 0-60% for 0.1 min, 75% for 5 min, 80% for 10 min, 90% for 10 min and 99% . ≪ / RTI > At 99%, the mobile phase B was maintained for 20 minutes to wash the column and then 0% for 10 minutes for column re-equilibration. The ESI voltage for the ion trap MS was set at 3.0 kV. The m / z range of the precursor scan MS was 400-1600 and the data-dependent CID analysis was performed at 40% normalized collision energy. The molecular structure of the lipids was determined by the LiPilot software, a fractional ion spectrum of lipid molecules, developed by the laboratory and confirmed by manual review.

SRM-based quantitation of methylated lipids was performed under different assay conditions. The lipid extracts of DU145 cell lines were treated with H-labeled methylation and the extracts of D-allose-treated cells with D-labeled methylation. The methylated lipid samples were mixed at a 1: 1 (H / D) ratio (10 [mu] g / l for each lipid sample) and nUPLC-ESI-MS / MS analysis was performed. The lipid mixture (2 [mu] l, 10 [mu] g) was loaded onto the same analytical column used for non-target identification and mobile phase A was used at a flow rate of 700 nL / min for 10 min. After sample loading, the flow rate of the column could be adjusted to 300 nL / min by switching the switching valve on and increasing the pump flow rate to 14 μl / min. Gradient elution II was initiated by increasing mobile phase B to 70% for 0.1 min, to 80% for 5 min, and finally to 100% for 10 min. Thereafter, the column was kept at 100% for 10 minutes for washing and 0% for 5 minutes. Detection of methylated PL was performed with a scan width of m / z 1.0 and a scan time of 0.001 s at 3.0 kV ESI in cation mode. The SRM quantification of methylated lipids was performed at 25 V CID for all species. For CL species with m / z value> 1500, SRM quantification was performed using ion trap MS. To evaluate the results obtained in the ILM method, conventional quantitation experiments were performed by analyzing individual lipid extracts with an internal reference set (0.5 pmol / [mu] l) with an odd acyl chain. Relative changes in each individual lipid molecule were analyzed by calculating the ratio of the corrected peak areas of the two samples (relative to the peak area of each standard).

2. Results and Discussion

(1) nUPLC-ESI-MS ⁿ analysis of methylated PL

Methylation was performed on mixtures of PL standards with different headgroups: PG, PI, PS, PA, PIP _n (PIP molecules with different degrees of phosphorylation in the inositol group), CL and Cer1P. The methylation of the phosphate group improved the ionization efficiency by increasing the hydrophobicity of the PL because the hydrophobic molecules tend to be easily separated from other interfering compounds in the ESI by the tendency to stay on the surface of the droplet, Respectively. In addition, the increase in hydrophobicity caused a longer retention time in the reversed phase column, and as a result, the material eluted under conditions with a higher proportion of organic solvent was more advantageous in generating charged droplets. Therefore, it has been necessary to investigate the influence of the ionizing agent added to the mobile phase of LC. In this experiment, three ionization media were tested by comparing their MS signal intensities using 29 PL standard material post H-methylation. (5 mM AF + 0.05% AH), 5 mM ammonium acetate (AA) and 5 mM AF used for MS analysis of methylated phosphoinositides, which were used as universal mediators in both cation and anion modes The effect of medium on the MS signal intensity of methylated PL is shown in FIG. Most PLs were detected as protonated forms ([M + H] ⁺ ). However, PG, PI, PA and CL were mainly detected as ammonium adduct ions ([M + NH ₄ ] ⁺ ). They have also been detected in their protonated form ([M + H] ⁺ ), but can be considered negligible. As can be seen in FIG. 6, 5 mM AF showed the highest ionization efficiency (up to 2-fold) for all PL groups including PIP _n molecules. Thus, 5 mM AF was used as the ionization vehicle of the mobile phase solution for further analysis.

The retention time and the ionization efficiency of polar PL were increased by increasing hydrophobicity of PL at methylation. Figure 3 shows 1) a mixture of 18 complete PL reference materials (1 pmol each) obtained in anion mode using 0.05% AH added to the mobile phase and 2) cation mode with 5 mM AF obtained by nUPLC-ESI-MS / MS (EIC) of the H-methylated PL reference material. 3) was obtained using a 1: 1 mixture of H-methylated and D-methylated PL standards (1 pmol each). The retention time of PL was increased as a result of methylation (2 in Fig. 3)). However, the signal intensities increased with the widths of the peaks numbers 13 to 18 being significantly reduced: in the case of intact, 0.25 min versus H-methylated (14: 0) ₄ -CL (peak number 18) 0.09 minutes. The degree of methylation depends on the number of polar head units of PL (1 for PI and PG, 2 for PS, PA and CL, 3 for PIP, 5 for PIP ₂ , 7 for PIP ₃ ) , Extended retention times were obtained in molecules with high methylation (Peak Nos. 1, 4, 6, 8 and 18) as compared to intact molecules. In addition, PIP _n molecules under intact conditions were not successfully detected in negative ion mode of MS due to the presence of additional phosphate groups in inositol (1 in FIG. 3). However, the methylation of the terminal phosphate group improved the detection efficiency of peaks 11 and 12 because of the increased hydrophobicity. In addition, the MS signal intensity was increased when the Cer1P (No. 10) and PA (No. 13 and 17) molecules were also methylated. It should be noted that the retention time was significantly increased at cardiolipin (CL) (peak number 18) after methylation but the detection was successful. CL analysis by MS with reversed-phase LC (RPLC) was relatively difficult due to the low retention time due to the presence of four acyl chains in the CL molecule as well as low ionization of the ESI due to the two phosphate groups. Methylation has great potential for selective profiling of trace amounts of CL specifically found in mitochondria. Since mitochondrial dysfunction is a prelude to neurodegenerative disorders, an efficient analysis of CL can prove very useful in assessing lipid intervention in mitochondrial-related diseases. Enhancement of CL profiling of cell extracts will be discussed in detail later.

The molecular structure of the H- and D-methylated PL can be distinguished on the basis of differences in the precursor ion m / z values, along with the data-dependent CID fraction ion spectra obtained by MS / MS / MS (MS ³ ). The spectra of the H- and D-methylated PL molecules obtained by nUPLC-ESI-MS ⁿ analysis are shown in FIG. The precursor ion MS spectra of peak number 13 (2 in FIG. 7) and 16: 0/18: 1-PA at t _r = 26.05 min in FIG. 7a) The form of the ammonium adduct in the form of the ammonium adduct at 720.5 ([M _H + NH ₄ ] ⁺ , subscript H for H-methylation and 724.5 ([M _D + NH ₄ ] ⁺ for D-methylation) Lt; RTI ID = 0.0 > labeled < / RTI > In Figure 7b, c each MS / MS spectrum _{[M H -PO 2 (OCH 3} ) 2] + and _{[M D -PO 2 (OCHD 2} ) 2] ⁺ as a significant and common having the same m / z 577.5 Shows loss of NH ₃ at m / z 703.6 ([M _H + NH ₄ -NH ₃ ] ⁺ ) and m / z 707.7 ([M _D + NH ₄ -NH ₃ ] ⁺ ) with formation of fraction ions. Figure 7d shows the MS ³ spectrum of the common ion m / z 577.5 in Figures 7b and c, with m / z 321.4 ([M _H -PO ₂ (OCH ₃ ) ₂ -R ₁ COOH] ⁺ ) and m / z 295.4 Loss of carboxylic acid in [M _H -PO ₂ (OCH ₃ ) ₂ -R ₂ COOH] ⁺ ), detection of acyl chain in the form of an acylium ion ([RCO] ⁺ ) at m / z 239.3 and 265.3, and ([RCO-H ₂ O] ⁺ ) from the acylium ion at m / z 211.3 and 247.3. Based on the CID spectrum using characteristic fraction ions, they can be readily identified by H-methylated and D-methylated 16: 0/18: 1-PA molecules.

(2) Efficiency of methylation

The yield of the methylation reaction was increased in the acidic condition (pH = 1 ~ 2) obtained by adding HCl. The methylation efficiency (%) of 24 lipid standard materials with and without addition of HCl was compared in Table 2. Methylation efficiency represents the percentage of methylated species relative to the initial intact PL species. The amount of methylated species was calculated by subtracting the peak area (relative to the internal standard of each specific PL species) of the precursor ion of the non-methylated species after methylation from the peak area of the same molecule without methylation. As the ionization efficiencies of the methylated and non-methylated species in the ESI are different due to the increased hydrophobicity of the methylated species, a comparison of the peak areas of the molecules reveals that the intact PL molecules after methylation in the anion mode of nUPLC-ESI-MS / Of the total. As shown in Table 2, the methylation efficiency of PL under acidic conditions was 96% or more in most species except for LPI molecules (~ 92.4%), while 75% to 94% efficiency was obtained under neutral conditions. The efficiency data for Cer1P and PIP _n are not included in Table 2 because these molecules are not analyzed by reversed phase nUPLC-ESI-MS / MS. However, these molecules were well detected after methylation. According to the literature, the methylation efficiency of Cer1P and PIP _n is reported to be 93.8 ± 0.2%. In this particular method, the methylation efficiency is based on the calculation of the remaining intact PL molecules, but it is uncertain whether all the molecules have been methylated. This was further investigated by measuring the relative proportions of the H- and D-methylated standards mixture while changing the mixing ratio.

(3) ILM-based quantification of PL by nUPLC-ESI-MS / MS

ILM-based quantification by nUPLC-ESI-MS / MS analysis was performed for the quantification of 29 methylated PL reference material pairs (H- and D-methylation). The H- and D-methylated PL standards were prepared in five different ratios: 0.25 (2: 8), 0.67 (4: 6), 1.00 (5: 5), 1.50 ) H / D and quantified by nUPLC-ESI-MS / MS according to the selective reaction monitoring (SRM) method. For SRM quantification, the type of quantification ion for each PL species was selected from the first MS / MS spectrum of the reference species of each lipid species. The left side of FIG. 4-1 shows the correlation between the calculated peak area ratio (H / D) of H- / D-methylated 16: 0/18: 1-PS and the mixing ratio obtained by nUPLC-ESI-MS / And exhibits a good linear relationship between H / D of 0.25 to 4.00. The right side of Figure 4-1 shows the overlapping extracted ion chromatogram (EIC) of H- and D-methylated PS molecules (triplicate of each sample) with an average H / D value of 4.19 +/- 0.17, ) By about 5%. The retention times of each species were 14.96 ± 0.04 for both H- and D-methylated PS, and the deviations were negligible. The ratio of the calculated peak area (H / D) of the 24 methylated PL species is shown in Table 3. Most of the PLs showed a reasonable H / D ratio in the experiment compared to the blending ratio of 6.6% or less relative to the average, but only the PI containing LPI showed an average of 42.6% in the experimental H / D ratio from the expected value, . This difference can be attributed to the steric hindrance caused by the cyclic ring of the inositol head group, which is relatively large compared to other head groups of PL. This indicates that the methylation of PI molecules is not as efficient as other PL molecules. However, PIP molecules with different degrees of phosphorylation exhibited a good correlation (within 6.14% error for the mean in Table 3) with expected H / D values. In PIP, PIP ₂ and PIP ₃ , the methylation of the glycerol skeleton at the phosphate group and the methylation of the terminal phosphate group at the inositol head group (two methylations per terminal phosphate group) were successfully performed.

The matrix effect on methylation-based quantitation was assessed by adding an odd acyl chain PL standard to the cellular lipid extract of DU145 cells and then treating the same aliquot of the mixture for H- and D-methylation. The respective H- and D-methylated PL standards contained in the cellular lipid extracts were mixed in the same manner with varying mixing ratios (H / D of 0.25, 0.67, 1.00, 1.50 and 4.00). The resulting mixture was analyzed as described above. According to the calculated ratios of the H / D methylated species, the deviation from the expected ratio was less than 6.3% on average for most species and nearly similar to the observed mean deviation in Table 3. This suggests that the matrix effect of the cell extract is not large.

(4) ILM-based quantification of PL extracts of HRPC cells by nUPLC-ESI-MS / MS

The ILM method was applied for the relative quantification of PL extracted from HRPC DU145 cells with or without D-allose treatment. PL extracts of D-allose treated cells were D-methylated and whole cell extracts were H-methylated. Two methylated products of the same aliquot were mixed together for nUPLC-ESI-MS / MS analysis. ILM-based quantitation of cell PL was performed only for PG, PS, PA and CL classes. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were not included in the ILM-based quantitation because the methylation at the primary amine group of PE formed PC molecules, making it almost indistinguishable from the original PC molecule of the cell extract It can not be.

A total of 112 species were identified (1 LPG, 33 PG, 1 LPS, 14 PS, 2 LPA, 18 PA and 43 CL). Since SRM does not distinguish the isomeric structure of the acyl chains, except PA molecules, the quantification was performed on 83 PLs. Of the 112 species, only 8 PA and 25 CL were identified from methylated PL according to the calculated peak area ratio (D / H) for PL species, along with the identified molecular structure of the isomeric form by the CID experiment , Which were not detected in intact PL extracts using the conventional method in negative ion mode and these results indicate that the detection limit was improved when analyzed after methylation of PA and CL. In particular, the identified number of CLs increased to 43 from methylated extracts, but only 18 from the complete PL extracts were identified. This is a significantly improved result compared to the reported number of identified CL (24 from PC-3 cells and 28 from the liver using high-resolution LC-MS). The improvement in CL detection is believed to be due to the increased ionization of methylated CL molecules due to increased hydrophobicity. Also, the determination of the four acylchain positions in the intact CL, which was otherwise difficult, was followed by the addition of each pair of acyl chains, (16: 0,16: 1), (18: 1, 18: 1) -CL and 16: 1, 18: 1), (16: 0, 18: 1) -CL. According to a series of CID spectra of methylated CL of cell extracts, the molecular structure of each set of two acyl chains is shown in the MS ³ experiment of the fraction ion ([R ₁ CO ₂ CH ₂ (R ₂ CO ₂ ) CHCH ₂ ] ⁺ ) Can be clearly distinguished and was easily obtained from the MS ² experiment of ammonium adducts of methylated cardiolipin molecules. As far as we know, this is the first report to demonstrate the confirmation of the position of the acyl chain in each glycerol molecule of CL in cationic mode. For the evaluation of the ILM-based quantitation, the measured D / H values were compared with the values obtained by the conventional quantitation method. In the conventional method, the intact PL extracts (with or without D-allose treatment) Was measured with a calibrated peak area relative to an internal standard (IS) specific to each head. Comparisons of the two quantitation methods were performed on 4 PGs, 4 PSs, 10 PAs, and 8 CLs that did not contain the isomer chain structure by the Student's test because the ILM- Depends on the total number of double bonds, whereas conventional quantitation methods depend on individual isomeric molecules. The p-value calculated from the two methods was found to be greater than 0.05 in all samples except 18: 1/18: 1-PS, indicating that the ILM-based quantitation method is not statistically different from the conventional method.

Other results indicate that, upon treatment with D-allose, most PS, PA and CL species decreased, while some PGs increased. There was a paper showing a change of more than 30% in D / H ratio. Reduced amounts of PS and PA could be attributed to the anti-proliferative effect of D-allose on DU145 cells and may be the result of mitochondria-mediated cell death pathways. The decreasing tendency of PA and CL and the increase in the level of PG are synthesized from PG by CL-producing enzyme in the presence of CL cytidine diphosphate (CDP) diacylglycerol and by using cytidine triphosphate (CTP) as a precursor, PA Cystyllyl-transferase was formed from PA by the enzyme. Thus, a marked reduction in PA amount after D-allose treatment (2 to 3 times for all 19 PAs except 16: 0-LPA) can be seen as a result of inhibiting the conversion of PG to CL.

3. Conclusion

In this example, the ability of the ILM-based quantification method was evaluated in cationic mode using an optimized ionization medium, 5 mM AF. This method improves the efficiency of the ESI and increases the hydrophobicity of the PL, thereby prolonging the retention time. The modified retention times of the H- and D-methylated lipids were approximately the same, demonstrating that PL can be analyzed simultaneously without internal reference material from a mixture of two different samples. The methylation efficiency was higher than 96% in most PL except for LPI (~ 92.4%) under acidic conditions. A good linear relationship was observed between H- and D-methylated PL with an error value of less than 6.6%. The ILM-based quantitation method was compared to the conventional method using internal standards in all kinds of PLs and the p-value of the Student's test, which was greater than 0.05 for all kinds of lipids except 18: 1/18: 1-PS , It was found to be equally effective. The prominent advantages of the ILM-based method include the detection of additional PLs (8 PAs and 25 CLs) that were not detected from intact PL extracts, improved CL analysis by increasing the detection capacity of methylated CLs, It is possible. As CL is specifically found in mitochondria, ILM-based quantitation methods can be applied, in particular, to geochronological analysis of mitochondrial-related diseases. The current method was not applied to PC and PE, but as a result of using C ¹³ labeled TMSD, the methylated PE had an m / z value of 3 Da greater than the intact PC with the same acyl chain structure because PE Is known to be made by a combination of three methylations in the primary amine or two methylations in the primary amine and methylation in the phosphate group.

Claims (10)

An extraction step of extracting lipids in vivo;
An alkylation step of introducing an iso-substituted alkyl group into the extracted lipid; And
An in vivo lipid assay method comprising an analytical step of analyzing alkylated lipids using a liquid chromatography-electrospray ionization-mass spectrometer system. The method according to claim 1,
Wherein the isotope is deuterium. The method according to claim 1,
Wherein the alkylation is methylation. The method according to claim 1,
Wherein the lipid is a phospholipid. 5. The method of claim 4,
A method for analyzing lipid in living body, wherein methylation is carried out on the phosphate group of the lipid. The method according to claim 1,
Wherein at least one of MEOD, DCI and D ₂ O is used in the extraction step and the alkylation step. The method according to claim 1,
Characterized in that in the analysis step a mobile phase comprising an ionization mediator is used. 8. The method of claim 7,
Wherein the ionization medium is at least one selected from the group consisting of ammonium formate, ammonium hydroxide, and ammonium acetate. The method according to claim 1,
Liquid chromatography-electrospray ionization-mass spectrometer system comprises a pump; Analytical column; valve; Metal wire; A microcross connected to the pump, the analysis column, the valve and the metal wire, respectively; And a pressure capillary connected to the valve. 10. The method of claim 9,
Wherein the pressure capillary is controlled during the elution of the lipids in the analysis step, and the mobile phase is flowed at a rate of 300 to 500 nL / min toward the analysis column and the mass spectrometer.

Images