JP7306676B2 - Lipid profiling system, lipid profiling method, and lipid profiling program - Google Patents

Description

The present invention relates to a lipid profiling system, a lipid profiling method, and a lipid profiling program.

Fatty acid side chains of lipids can be broadly classified into three main types: saturated fatty acids, unsaturated fatty acids, and highly unsaturated fatty acids. Each lipid class determines the main compound to some extent. In addition, although highly unsaturated fatty acids are hardly present in diglycerides (DG) used as precursors for biosynthesis of each glycerophospholipid, their proportion is greatly increased in glycerophospholipids. Such an increase in the ratio of polyunsaturated fatty acids is very important in considering the quality of lipids in vivo. Recent advances in the sensitivity of mass spectrometers have made it possible to qualitatively characterize the fatty acid side chains of these glycerophospholipids and measure their ionic strength.
For example, Patent Literature 1 describes a mass spectrometry system that identifies lipids from mass spectra.

JP-A-2006-226730

Quantitative values of lipid molecules are generally calculated by preparing an external calibration curve using chemically synthesized standard products. However, since it is practically difficult to obtain standards for all lipid molecules, comprehensive quantification of lipid molecules has not been achieved. In addition, since the product ionic strength of lipid molecules with two or more fatty acid side chains differs depending on the lipid molecule, it is possible to compare the amounts of the same lipid molecules, but it is not possible to compare the amounts of different lipid molecules. .

The present invention has been made in view of the above circumstances, and a lipid profiling system, a lipid profiling method, and a lipid that can calculate the quantitative ratio between different lipid molecules having two or more fatty acid side chains. The purpose is to provide a profiling program.

In one embodiment of the present invention, the type of fatty acid side chain and the ionic strength of the fatty acid side chain obtained by tandem mass spectrometry of a lipid having two or more fatty acid side chains are determined from a plurality of groups belonging to the same lipid class. an acquisition unit that acquires each type of lipid, the product ionic strength of the first type of fatty acid side chain possessed by the first type of lipid, and the product ionic strength of the first type of fatty acid side chain possessed by the second type of lipid; and a lipid amount ratio calculator that calculates the amount ratio between the first type of lipid and the second type of lipid based on the ratio of .

In one embodiment of the present invention, in the lipid profiling system described above, the first type of lipid has a first type of fatty acid side chain and a second type of fatty acid side chain, and the lipid amount ratio calculation unit is , a first correction coefficient indicated by the ratio of the product ionic strength of the first type of fatty acid side chain of the first type of lipid and the product ionic strength of the second type of fatty acid side chain; said quantitative ratio between one type of lipid and said second type of lipid; the product ionic strength of said second type of fatty acid side chain possessed by said first type of lipid; and said calculating the quantitative ratio between the first type of lipid, the second type of lipid, and the third type of lipid based on the product ionic strength of the second type of fatty acid side chain and the ratio of .

In one embodiment of the present invention, in the lipid profiling system described above, the second type of lipid has the first type of fatty acid side chain and the third type of fatty acid side chain, and the lipid amount ratio calculation unit is further a second correction factor indicated by the ratio of the product ionic strength of the first type of fatty acid side chain and the product ionic strength of the third type of fatty acid side chain of the second type of lipid; and , the quantitative ratio between the first type of lipid and the second type of lipid, the product ionic strength of the third type of fatty acid side chain possessed by the second type of lipid, and the fourth type of lipid The quantitative ratio between the first type of lipid, the second type of lipid, and the fourth type of lipid based on the ratio of the product ionic strength of the third type of fatty acid side chain having Calculate

In one embodiment of the present invention, in the lipid profiling system described above, the lipid amount ratio calculation unit further includes the said first type of lipid and said second type of lipid based on the quantitative ratio and said quantitative ratio between said first type of lipid, said second type of lipid and said fourth type of lipid and the quantitative ratio between the third type of lipid and the fourth type of lipid.

In one embodiment of the present invention, in the lipid profiling system described above, the concentration acquisition unit acquires the concentration of the first type of lipid, the concentration of the first type of lipid acquired by the concentration acquisition unit, A lipid concentration calculation unit that calculates the concentration of the second type of lipid based on the quantitative ratio between the one type of lipid and the second type of lipid.

In one embodiment of the present invention, in the lipid profiling system described above, the lipid concentration calculation unit calculates the concentration of the first type of lipid acquired by the concentration acquisition unit, the concentration of the first type of lipid and the second type of lipid. calculating the concentration of the second type of lipid and the concentration of the third type of lipid based on the quantitative ratio between the lipid of and the third type of lipid.

In one embodiment of the present invention, in the lipid profiling system described above, the lipid concentration calculation unit calculates the concentration of the first type of lipid acquired by the concentration acquisition unit, the concentration of the first type of lipid and the second type of lipid. calculating the concentration of the second type of lipid and the concentration of the fourth type of lipid based on the quantitative ratio between the lipid of and the fourth type of lipid.

One embodiment of the present invention is the lipid profiling system described above, wherein the lipid classes are diacylglycerols, triacylglycerols, phosphatidylcholines, phosphatidylethanolamines, phosphatidic acids, phosphatidylserines, phosphatides. selected from the group consisting of dilinositol and phosphatidylglycerol;

One embodiment of the present invention is a step of analyzing a plurality of types of lipids belonging to the same lipid class, which are lipids having two or more fatty acid side chains, with a tandem mass spectrometer, and , obtaining the type of the fatty acid side chain and the product ionic strength of the fatty acid side chain for each type of the lipid; and the product ionic strength of the first type of fatty acid side chain possessed by the first type of lipid; calculating the quantitative ratio between the first type of lipid and the second type of lipid based on the ratio of the product ionic strength of the first type of fatty acid side chain possessed by the second type of lipid; and a method of lipid profiling.

One embodiment of the present invention provides a computer with lipids having two or more fatty acid side chains, which are obtained by tandem mass spectrometry for a plurality of types of lipids belonging to the same lipid class, the types of the fatty acid side chains, and obtaining the product ionic strength of the fatty acid side chain for each type of the lipid; calculating the quantitative ratio between the first type of lipid and the second type of lipid based on the ratio of the product ionic strength of one type of fatty acid side chain and A lipid profiling program.

According to the present invention, a lipid profiling system, a lipid profiling method, and a lipid profiling program are provided that are capable of calculating quantitative ratios between different lipid molecules having two or more fatty acid side chains.

It is a figure explaining the classification of a lipid. It is a figure explaining the kind of fatty acid side chain. BRIEF DESCRIPTION OF THE DRAWINGS It is a figure explaining the outline|summary of tandem mass spectrometry. (A) is a diagram explaining the outline of a tandem mass spectrometer, and (B) is a schematic diagram of lipid fragmentation by tandem mass spectrometry. It is a figure showing an example of composition of a lipid profiling system concerning one embodiment of the present invention. FIG. 4 is a diagram showing an example of the operation of the lipid profiling system according to one embodiment of the present invention; FIG. 4 is a diagram showing an example of the operation of the lipid profiling system according to one embodiment of the present invention; FIG. 4 is a diagram showing a specific example of the operation of the lipid profiling system according to one embodiment of the present invention; It is a figure which shows an example of the verification result of the lipid profiling system concerning one Embodiment of this invention. MRM conditions used in specific examples of lipid profiling are shown. Correction factors for each fatty acid side chain in each lipid class calculated in the specific example of lipid profiling are shown. A specific example of lipid profiling shows the results of analyzing a normal liver sample and a nonalcoholic steato-hepatitis (NASH) sample. A specific example of lipid profiling shows the results of analyzing normal brain samples and nonalcoholic steato-hepatitis (NASH) mouse brain samples. A specific example of lipid profiling shows the results of analyzing a normal liver sample and a fatty liver sample. A specific example of lipid profiling shows the results of analyzing normal brain samples and nonalcoholic steato-hepatitis (NASH) mouse brain samples.

[Lipid]
FIG. 1 is a diagram explaining the classification of lipids. Lipids include simple lipids that are esters of alcohol and fatty acids, complex lipids that contain phosphoric acid and sugar in the molecule, and derived lipids that are derived from simple lipids and complex lipids by hydrolysis. ).
Simple lipids are classified as having a glycerol backbone, a sterol backbone or a ceramide backbone. Lipids with a glycerol backbone fall into the monoacylglycerol (MG), diacylglycerol (DG), and triacylglycerol (TG) lipid classes.
Complex lipids are classified into glycerophospholipids and sphingolipids. Glycerophospholipids include phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylserine ( PS) and other lipid classes.

FIG. 2 is a diagram illustrating types of fatty acid side chains of lipids. Fatty acid side chains are classified into side chains composed of saturated fatty acids and side chains composed of unsaturated fatty acids.

The lipid profiling system according to embodiments of the present invention is applicable to lipid classes with two or more fatty acid side chains (DG, TG, PC, PG, PI, PE, PA, PS, etc.).

[Tandem mass spectrometry]
FIG. 3 is a diagram showing an outline of tandem mass spectrometry. FIG. 3A is a diagram showing an outline of a tandem mass spectrometer that implements tandem mass spectrometry. Mass spectrometry (MS) is an analytical method in which compounds in a sample are gaseously ionized, separated according to m/z (mass-to-charge ratio), and the ion intensity of each ion is measured. A tandem mass spectrometer (MS/MS) is a device with two mass spectrometers coupled in series with a collision chamber in between. As shown in FIG. 3(A), in the ionized sample, ions with a specific m/z value (precursor ions) are selected in the first mass spectrometer (Q1) and fragmented ( fragmentation). Fragment ions (product ions) generated by fragmentation of precursor ions are separated according to m/z values in the second mass spectrometer (Q3) and detected by a detector.

FIG. 3(B) shows a schematic representation of fragmentation in diacylglycerol (DG) and phospholipids (PC, PE, PS, PI, PG).

[Implementation]
The lipid profiling system 10 of this embodiment will be described below with reference to the drawings.
FIG. 4 is a diagram showing an example of the functional configuration of the lipid profiling system 10 of this embodiment. This lipid profiling system 10 calculates the amount ratio LR and concentration CO between lipids belonging to the same lipid class in a sample.

In FIG. 4, the tandem mass spectrometer 20 analyzes a sample containing lipids L1 to Ln, and measures the product ion intensity of each fatty acid side chain of each lipid.
A plurality of types of lipids L1 to Ln belong to the same lipid class LC, have a common head group HG, and have two or more fatty acid side chains FC. Lipid L1 has a fatty acid side chain α and a fatty acid side chain x as fatty acid side chain types L1(k1) and L1(k2). Lipid L2 has a fatty acid side chain α and a fatty acid side chain y as fatty acid side chain types L2(k1) and L2(k2). Lipid L3 has a fatty acid side chain z and a fatty acid side chain x as fatty acid side chain types L3(k1) and L3(k2). Lipid Ln has fatty acid side chain z and fatty acid side chain y as fatty acid side chain types Ln(k1) and Ln(k2).

In the tandem mass spectrometer 20, the precursor ions generated by the ionization of the lipids L1 to Ln are separated according to the m/z value in the first mass spectrometry section (Q1), and the separated precursor ions are separated in the collision chamber (Q2). It is fragmented to produce product ions L1(p1)-L1(p2) derived from each fatty acid side chain of lipids L1-Ln, respectively. In the second mass spectrometer (Q3), the product ions L1(p1) to L1(p2) are separated according to the m/z value, and the ion intensities L1(i1) to Ln(i2) of each product ion are detected by a detector (not shown). is detected. Fatty acid side chain types L1(k1) to Ln(k2) are identified based on the respective m/z values of product ions L1(p1) to L1(p2). That is, the lipid L1 has the fatty acid side chains α and x as the fatty acid side chain types L1 (k1) and Ln (k2) based on the m/z values of the product ions L1 (p1) and L1 (p2). identified as having (L1(α), L1(x)). Similarly, the types of fatty acid side chains are identified for lipids L2-Ln.

The types of fatty acid side chains and the product ionic strengths L1 (k1, i1) to Ln (k2, i2) of the lipids L1 to Ln identified as described above are input to the lipid profiling system 10, and the lipid profiling system 10 The quantity ratio LR between the lipids L1-Ln and the concentrations CO1-COn of the lipids L1-Ln are calculated.

[Functional configuration and operation of lipid profiling system 10]
A specific functional configuration and operation of the lipid profiling system 10 will be described.
The lipid profiling system 10 includes an acquisition unit 110 , a lipid amount ratio calculation unit 120 , a lipid concentration acquisition unit 130 , a lipid concentration calculation unit 140 , an output unit 150 and a storage unit 160 . In this example, the lipid profiling system 10 is described as including the storage unit 160, but the present invention is not limited to this. For example, the lipid profiling system 10 and the storage unit 160 may be configured as separate devices, such as the storage unit 160 being realized by a cloud server or the like.

FIG. 5 is a diagram showing an example of the operation of the lipid profiling system 10 of this embodiment. An example of the operation of the lipid profiling system 10 will now be described with reference to FIGS. 4 and 5. FIG. In the following example, the case of determining the quantitative ratio of three types of lipids L1 to L3 will be described, but it is not limited to this, and the quantitative ratio between lipids can be similarly determined for any lipid of lipids L1 to Ln. can be done.

(Step S10)
The acquisition unit 110 obtains the fatty acid side chain types L1 (k1) to L3 (k2) and the fatty acid side chain types L1 to L3 (k2) obtained by tandem mass spectrometry of lipids L1 to L3 having two or more fatty acid side chains FC. Product ionic intensities L1(i1) to L3(i2) are obtained for multiple types of lipids L1 to L3 belonging to the same lipid class LC, respectively.

The fatty acid side chain types L1(k1) to L3(k2) acquired by the acquisition unit 110 may be m/z values of the product ions L1(p1) to L3(p2). In this case, the storage unit 160 may store a fatty acid type reference table that refers to fatty acid types L1(k1) to L3(k2) from each m/z value of product ions.

The fatty acid side chain types L1(k1) to L3(k2) and the product ionic strengths L1(i1) to L3(i2) of the fatty acid side chains acquired by the acquisition unit 110 are obtained from other sources connected to the lipid profiling system 10. Supplied from a device or the like. Other devices connected to the lipid profiling system 10 include, for example, the tandem mass spectrometer 20, a host computer device of the lipid profiling system 10, and an operation device such as a keyboard or touch panel of the lipid profiling system 10 ( not shown).
The acquisition unit 110 outputs the acquired fatty acid side chain types L1 (k1) to L3 (k2) and the product ionic intensities L1 (i1) to L3 (i2) of the fatty acid side chains to the lipid amount ratio calculation unit 120.

(Step S20)
The lipid amount ratio calculator 120 calculates the product ionic strength L1(iα) of the first type of fatty acid side chain α of the first type of lipid L1 and the first type of fatty acid side chain α of the second type of lipid L2. and the product ion intensity L2(iα) of .

The lipid L1 has a fatty acid side chain α as the type L1(k1) of the fatty acid side chain, and its product ionic strength L1(i1) is L1(iα). The lipid L2 has a fatty acid side chain α as the fatty acid side chain type L2(k1), and its product ionic strength L2(i1) is L2(iα). The lipid amount ratio calculator 120 calculates the product ionic strength ratio (L2(iα)/ L1(iα)) is calculated.

(Step S30)
The lipid amount ratio calculation unit 120 calculates the product ionic strength ratio of the fatty acid side chain α between the lipids (L1, L2) having the fatty acid side chain α (L2(iα)/L1(iα) ), the quantitative ratio LR(α) between the first type of lipid L1 and the second type of lipid L2 is calculated.

The product ionic strength ratio of the same fatty acid side chain α generated from lipids L1 and L2 belonging to the same lipid class LC is proportional to the quantitative ratio of lipid L1 and lipid L2. Therefore, the quantitative ratio LR (L1, L2) between the lipids L1 and L2 can be calculated from the product ionic strength ratio (L2(iα)/L1(iα)) of the fatty acid side chain α. That is, it can be calculated as LR(L1, L2)=L2(iα)/L1(iα).

(Step S40)
The first type of lipid L1 has a fatty acid side chain α as the first type of fatty acid side chain L1(k1), and its product ionic strength L1(i1) is L1(iα). The lipid L1 further has a fatty acid side chain x as the second type of fatty acid side chain L1(k2), and its product ionic strength L1(i1) is L1(ix).
In step S40, the lipid amount ratio calculator 120 calculates the product ionic strength L1(iα) of the first type fatty acid side chain α of the lipid L1 and the product ionic strength L1(ix) of the second type fatty acid side chain x. , and a first correction coefficient CF(α, x) is calculated.
The correction factor CF (α, x) is the ratio of the product ionic strength L1 (ix) of the fatty acid side chain x to the product ionic strength L1 (iα) of the fatty acid side chain α in the lipid L1 (L1 (ix) / L1 (iα) ).

(Step S50)
The lipid amount ratio calculation unit 120 calculates the correction coefficient CF (α, x) calculated in step S40, the amount ratio LR (L1, L2) between the lipid L1 and the lipid L2 having the fatty acid side chain α, and the lipid L1 has The ratio of the product ionic strength L1 (ix) of the second type of fatty acid side chain x to the product ionic strength L3 (ix) of the fatty acid side chain x of the third type of lipid L3 (L3 (ix) / L1 (ix) ), and the quantitative ratio LR (L1, L2, L3) between the lipid L1, the lipid L2, and the lipid L3.

(Step S55)
When the fatty acid side chain L2 (k2) of the lipid L2 and the fatty acid side chain L3 (k1) of the lipid L3 are the same type of fatty acid side chain (that is, y = z), the fatty acid side chains α and y of the lipid L2 A correction coefficient CF (α, y) indicated by the ratio of the product ion intensities L2 (iα) and L2 (iy) is calculated, and the correction coefficient CF (α, y) and the quantity ratio LR (L1, L2) are calculated. , the product-in intensity ratio (L3(iz)/L2(iy)) (where y=z), and the quantity ratio LR′(L1, L2 , L3) may be calculated. Next, the quantity ratio LR (L1, L2, L3) calculated in step 50 is compared with the quantity ratio LR' (L1, L2, L3). If the difference is equal to or greater than the threshold, the process may return to step S40 to adjust the values of the correction coefficient CF(α, x) and the correction coefficient CF(α, y). A maximum likelihood method may be used to adjust each correction coefficient.

(Step S60)
In step S60, the lipid ratio LR (L1, L2, L3) calculated in step S50 is output to the output unit 150, and the process ends.

In the above, the quantitative ratio between the three types of lipids, lipid L1, lipid L2, and lipid L3, was calculated. Acquire the type of fatty acid side chain and the product ionic strength L1 (k1, i1) to Ln (k2, i2) of each fatty acid side chain, and repeat steps similar to steps S20 to S50 between any three types of lipids. , the quantitative ratio between lipids L1 to Ln. For example, when the lipid Ln has fatty acid side chains z and y, the quantitative ratio LR (L1, L2) between the lipid L1 and the lipid L2 is calculated in steps S20 and S30, and in step S40, the lipid L2 The correction factor CF(α, y) may be calculated from the ratio of the product-in intensity of the fatty acid side chain α and the fatty acid side chain y. Next, in step 50, the correction factor CF (α, y), the quantitative ratio LR (L1, L2) between the lipid L1 and the lipid L2, and the product ionic strength L2 (iy) of the fatty acid side chain y of the lipid L2 Based on the ratio of the product ionic strength Ln (iy) of the fatty acid side chain y of the lipid Ln and the lipid The interval ratio LR (L1, L2, Ln) can be calculated. Furthermore, based on the respective quantitative ratios of lipid L3 and lipid Ln to lipid L1 and lipid L2, the quantitative ratio LR (L1, L2, L3, Ln) of lipid L1, lipid L2, lipid L3, and lipid Ln is calculated. can be done. Similarly, steps S20 to S50 may be repeated to calculate the quantitative ratios of the lipids L1 to Ln until the quantitative ratios among all the lipids L1 to Ln are calculated. A maximum likelihood method may be used to calculate the quantitative ratio between the lipids L1 to Ln.

The lipid profiling system 10 of the present embodiment may have a function of acquiring the concentration of lipid L1 and calculating the concentrations of lipids L2 to Ln.
FIG. 6 is a diagram showing an example of the lipid concentration calculation operation of the lipid profiling system 10 of the present embodiment. An example of the operation of the lipid profiling system 10 for calculating the lipid concentration will be described below with reference to FIGS. 4 and 6. FIG.

(Step S110)
The concentration acquisition unit 130 acquires the concentration CO1 of the first type of lipid L1. For example, a known amount of lipid L1 is added as a radiolabeled standard sample to the sample to be analyzed by the tandem mass spectrometer 20, and the added amount can be used as the concentration CO1 of the lipid L1.
The concentration CO1 of the lipid L1 acquired by the lipid concentration acquisition unit 130 is supplied from another device or the like connected to the lipid profiling system 10 . Other devices connected to the lipid profiling system 10 include, for example, the tandem mass spectrometer 20, a host computer device of the lipid profiling system 10, and an operation device such as a keyboard or touch panel of the lipid profiling system 10 ( not shown).
The lipid concentration acquisition unit 130 outputs the acquired concentration CO1 of the lipid L1 to the lipid concentration calculation unit 140 .

(Step S120)
The lipid concentration calculator 140 acquires the lipid ratio calculated in step S30 or step 50 from the lipid ratio calculator 120 .

(Step S130)
The lipid concentration calculation unit 140 calculates the concentration CO2 of the lipid L2 based on the concentration CO1 of the lipid L1 acquired by the concentration acquisition unit 130 and the quantitative ratio LR between the lipid L1 and the lipid L2. Alternatively, the lipid concentration calculation unit 140 calculates the concentration CO2 of the lipid L2 and the lipid Calculate the concentration CO3 of L3. Concentrations of the other lipids L4 to Ln can be similarly calculated from the ratio of the concentration CO1 to the lipid L1 and the lipid L1.

(Step S140)
In step S140, the concentrations of lipids L2 to Ln calculated in step S130 are output to the output unit 150, and the process ends.

[Specific operation example of lipid profiling system]
A specific operation example of the lipid profiling system of this embodiment will be described below.

FIG. 7 is a diagram illustrating a specific example of the operation of lipid profiling system 10 in a lipid profiling example. According to the flow chart on the left, a Quantity Ratio Table is created from the product ion intensity obtained by tandem mass spectrometry using the maximum likelihood method or the like. (A) shows a specific example of the Quantity Ratio Table.
Next, based on the Quantity Ratio Table, a virtual calibration curve is created. (B) and the bottom right graph show a specific example of a virtual calibration curve. The lower right graph shows a virtual calibration curve with the ionic strength of the fatty acid side chain (18:1) as the vertical axis. Here, the slope of the virtual calibration curve is set so that the difference between the measured value of the product ion intensity and the measured value of the product ion intensity is minimized while changing the quantitative value with a solver (least squares method). The reliability of the virtual calibration curve is determined by R ² , and if R ² is within a predetermined range (e.g., R ² > 0.94), proceed to "Calculate Distribution Ratio" in the left flowchart and A quantitative calibration curve is used to calculate the lipid ratio as in (C).
The slope of the virtual calibration curve corresponds to the correction coefficient CF described with reference to FIG.

Although the method for calculating the quantitative ratio of each lipid from the measured value of the product ion intensity of each lipid by tandem mass spectrometry is not particularly limited, for example, the maximum likelihood method can be used. An example of calculation of the lipid ratio by the maximum likelihood method is shown below.

FIG. 8 is a diagram illustrating a method of verifying a virtual calibration curve by ^R2 of the virtual calibration curve. If the quantified value of the lipid molecule is directly proportional to the product ion intensity of each fatty acid side chain, ^R2 converges to 1 as in (A). However, since it is believed that there is actually a measurement error or the like, the allowable line is defined as R ² >0.94 in the form of (B). When there are samples that are not in direct proportion (PC(18:0)(18:1)) as in (C), all calibration curves do not converge within 0.94. In (C), the calibration curve is drawn at (18:1) with R ² > 0.94, but at (18: 2) there is a large error of R ² < 0.94, and at (18: 0) there is no do not converge.

Although the embodiments of the present invention have been described above in detail with reference to the drawings, the specific configuration is not limited to these embodiments, and modifications can be made as appropriate without departing from the scope of the present invention. can.

Each of the devices described above has a computer inside. The process of each process of each device described above is stored in a computer-readable recording medium in the form of a program, and the above process is performed by reading and executing this program by a computer. Here, the computer-readable recording medium refers to magnetic disks, magneto-optical disks, CD-ROMs, DVD-ROMs, semiconductor memories, and the like. Alternatively, the computer program may be distributed to a computer via a communication line, and the computer receiving the distribution may execute the program.

Further, the program may be for realizing part of the functions described above.
Further, it may be a so-called difference file (difference program) that can realize the above-described functions in combination with a program already recorded in the computer system.

[Specific examples of lipid profiling]
Specific examples of lipid profiling using the lipid profiling system 10 of the present embodiment are shown below.

Materials and analysis methods used in specific examples of lipid profiling are as follows.

<Compounds and Reagents>
Distilled water (LC/MS grade) and methanol (LC/MS grade) were purchased from Kanto Kagaku Co., Ltd. (Tokyo, Japan). HPLC grade chloroform was purchased from Kishida Chemical Co., Ltd. (Osaka, Japan). SPLASH ^™ Lipidomix ^™ Mass Spec Standard including all major lipid classes (15:0/[D7]18:1-PA (7 ng), 15:0/[D7]18:1-PC (160 ng), 15: 0/[D7]18:1-PE (5 ng), 15:0/[D7]18:1-PG (30 ng), 15:0/[D7]18:1-PI (10 ng) and 15:0/ [D7] 18:1-PS (5 ng) per 1 μL Methanol) was obtained from Avanti Polar Lipid Inc.; (Alabaster, AL, USA). Ammonium acetate was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Carbon dioxide (99.5% grade,) was purchased from Yoshida Oxygen Co., Ltd. (Fukuoka, Japan) and used as the SFC mobile phase. DG (18:0/20:4), PE (18:0/20:4), PG (16:0/18:1), PS (18:0/18:1), PI (18:0/ 20:4), and 10 PCs from Avanti Polar Lipids Inc. purchased from

<Animal and diet treatment>
All animal care and testing protocols were approved by the Animal Ethics Committee of Kyushu University School of Medicine and were performed in accordance with the recommendations of the Kyushu University School of Medicine Committee for Care and Use of Laboratory Animals.

<MCD diet treatment>
Male C57BL6 mice, 5 weeks old, were purchased from Japan Charles River Co., Ltd. (Yokohama, Japan) and maintained at 24° C. with a 12 h/12 h light/dark cycle. An acute pathology model was developed by feeding mice a methionine-choline deficient (MCD) diet (Oriental Yeast Co., Ltd., Tokyo, Japan). Prior to testing, all animals were acclimated on normal diet for 1 week and allowed free access to tap water and appropriate diet (MF diet, Oriental Yeast Co., Ltd.). At 6 weeks of age, mice were divided into 2 groups (n=5) and maintained on normal chow or MCD diet for 2 weeks.

<Lipid extraction in mouse liver and brain>
A 20 mg mouse liver sample was placed in each 2 mL Eppendorf tube. To extract lipids from liver and brain, 1 mL of ice-cold methanol was added to the samples and sonicated for 1 minute to extract lipids. After centrifugation (16,000×g, 4° C., 1 minute), 500 μL of supernatant was collected and extracted by the Bligh-Dyer extraction method. A mixed solvent (MeOH:CHCl ₃ :H ₂ O=10:5:3 (v/v/v)) was added, vortexed for 1 minute, and then centrifuged (16,000×g, 4° C., 5 minutes). and remove impurities. After that, 300 μL of lower phase (chloroform phase) was collected in another glass tube. Finally, 300 μL of methanol was added to the tube, solution A was added and stored at −80° C. until analysis.

<SFC/QqGMS Analysis>
A Nexera UC system (SFC) (Shimadzu Corporation, Kyoto, Japan) equipped with an LCMS8060 QqQMS controlled by LabSolutions version 5.80 (Shimadzu Corporation, Kyoto, Japan) was used for lipid separation. A DEA column (100×3.0 mm id, ACQUITY UPC2™ Torus dimethylamine, Waters Corp.) was used for the separation. SFC conditions were as follows: mobile phase A, _SCCO2 ; mobile phase B, 95% methanol/5% water with 0.1% (w/v) ammonium acetate; back pressure, 10 MPa; °C; sample tray temperature, 5°C. The mobile phase was delivered to the column at a flow rate of 1.0 mL/min. The mobile phase gradient program was as follows: A/B = 100/0, followed by linear gradient to A/B = 30/70 for 15 min and held for 3 min, followed by linear gradient to A /B = 100/0 for 0.1 min, and terminating at A/B = 100/0 for 1.9 min. QqQMS, in which 2 μL of sample was injected with the autosampler, was performed after optimization. MRM conditions are shown in FIG.

<Optimization of MRM transition settings>
Identification of precursor ions of each of the six lipid classes (DG, PC, PE, PS, PG, PI) and analysis of fragmentation patterns were performed by SFC flow injection analysis in positive or negative ion mode. Precursor ions of each lipid were observed in positive ion mode ([M+H] ⁺ or [M+NH ₄ ] ⁺ ) and/or negative ion mode ([M−H] ⁻ or [M+CH3COO] ⁻ ) by addition of ammonium acetate. We optimized the MS collision energies for each lipid class of LCMS8060 based on the most abundant adduct ions. Figure 9 shows the optimization results of MS collision energies for six lipids and the MRM transition settings for seven fatty acid side chains. After generating all MRM transitions, the abundance ratio of monoisotonic ions was calculated. This is because the abundance ratio of monoisotonic ions varies depending on the fatty acid side chain.

<Compound annotation>
LabSolutions version 5.80 (Shimadzu Corporation, Kyoto, Japan) was used for the analysis of MRM spectral data. Furthermore, the data (lcd format) were converted to mzML format using ProteoWizard version 3. These data were then input into MRMPROBS version 2.26. Then, smoothing of MRM spectral data (smoothing method, linear weighted moving average; smoothing level, 5 scan; minimum peak width, 5 scan; minimum peak height, 100 amplitude) was performed using MRMPROBS.
Compounds were annotated essentially when all peaks of the compound's MRM transitions were at the retention time (RT). Using these data, the mean and standard deviation of the compounds in each sample were calculated using Excel 2013.

<Lipid profiling method>
In lipid classes with two fatty acid side chains (DG, PC, PE, PS, PI, PG), among the 28 compounds each, 7 compounds with the same two fatty acid side chains There are 21 types of compounds with different fatty acid side chains (28=7+21). Each of the 21 types of compounds with two different fatty acid side chains has two MRM transitions, and these two transitions differ in ionic strength depending on the type of fatty acid side chain, but the quantitative value is the same for one compound. Also, the ion intensity ratio between the two transitions is determined by the difference in fragmentation efficiency of the mass spectrometer and is constant regardless of concentration. Therefore, simultaneous equations can be constructed on the premise that the compound concentration and the product ion intensity are directly proportional to the quantitative values (unknowns) of the 21 kinds of compounds to be obtained at 42 kinds of ion intensities. According to the flowchart in FIG. 7, a virtual calibration curve with 6 calibration points was created from 7 types of fatty acid side chains for each lipid class from simultaneous equations. The correlation coefficient R ² of the virtual calibration curve was calculated, and if it was 0.99 or more, it was adopted as a formal correction coefficient as a reliable calibration curve, and the quantitative ratio was calculated using the calibration curve for all compounds. .
As a standard substance, an isotope-labeled (18:1) compound having a fatty acid side chain (Lipidomix Splash, Avanti Inc.) was mixed for each lipid class, and the ionic strength of the (18:1) fatty acid side chain was determined. Relative quantification (mg/ml) of each fatty acid side chain was performed using a calibration curve with 7 types of fatty acid side chains as a reference. The bar graph in FIG. 10 shows the correction factors for other fatty acid side chains, with the (18:1) fatty acid side chain as a reference value of 1.

<Comparison with standard product>
It is unknown to what extent the fatty acid side chains are bound to each of the sn-1 and sn-2 positions of the lipid. Table 1 shows the results of measurement at similar MRM transitions using prepared specimens. Since the fatty acid side chains bound to sn-1 and sn-2 of each sample are known in advance, the ionic strength ratio can be obtained with the type and bonding mode of the fatty acid side chains known in advance. When compared with the standard, the normal phospholipids of the measured samples are close in value to the standard with saturated fatty acids at sn-1 and unsaturated fatty acids at sn-2. This is particularly evident for PC (18:0/18:1) and PC (16:0/18:1) for which positional isomer standards were available.

<Results of lipid profiling>
The results of lipid profiling are shown in FIGS. 11 and 12. FIG. FIG. 11 shows the results of lipid profiling of normal liver and NASH (nonalcoholic steato-hepatitis) mouse liver. NASH has elevated lipid abundance in most lipid classes. Quantitative values of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) having only one fatty acid side chain are also shown for reference. In LPC and LPE, fatty acid side chains are MRM-measured in positive ion mode. Noteworthy is PG, and although PG as a whole has increased, palmitic acid (16:0) has not increased at all (and therefore decreased as a composition ratio). Given that NASH is a hepatic mitochondrial deficiency, changes in PGs present only in mitochondria may be important in studying NASH. Also, LPC and LPE are total values of only 6 kinds of fatty acid side chains, but the values of other phospholipids are decreased although they are increased.

FIG. 12 shows the results of lipid profiling on NASH mouse brain extracts. An increase in phospholipids and a slight decrease in LPC and LPE were confirmed, although not as marked as in the liver, but there was almost no change in the composition ratio of constituent fatty acids.

13 and 14 show detailed quantitative values for each compound. The total amount of all quantified compounds is shown in the upper part of the figure as a total amount, and the ratio of the breakdown is shown in the lower part of the figure.

<Discussion of lipid profiling results>
From the result of the correction factor of the ionic strength ratio of the fatty acid side chain of each lipid (Fig. 10), the neutral loss of the saturated fatty acid ((16:0), (18:0)) in the transition for quantification of DG and the highly unsaturated Comparing the neutral losses of fatty acids ((20:4), (22:6)), the ionic strength difference is 5 to 10 times or more. From these results, it can be said that both arachidonic acid and DHA are compounds that are very easily eliminated compared with other fatty acid side chains. There is an ionic strength difference of 6 to 20 times or more between the ionic strength of the saturated fatty acid and the ionic strength of the highly unsaturated fatty acid in the transition for quantitative determination of PS. In addition, in other glycerophospholipids, the ionic strength of the fatty acid side chain was measured in the same way as PS, but PC and PE show relatively similar trends, and both (18: 2) are easy to detach. . It was found that the ionic strength ratios of the same fatty acid side chains differ considerably depending on the type of glycerophospholipid. Overall, the transition of glycerophospholipids has a tendency opposite to the neutral loss of DG, and the ionic strength of polyunsaturated fatty acids, especially DHA, is low compared to other fatty acids. In addition, from the results of measuring samples with known sn-1 and sn-2 in advance (Table 1), it was found that the ionic strength was reversed between positional isomers of sn-1 and sn-2 even for the same fatty acid. I can ask. Based on this fact, with respect to lipids having two fatty acid side chains, the fatty acid side chain bound to sn-2 tends to be released very easily when mass spectrometry is used. In other words, it is thought that the product ionic strength also increased in parts other than the types of fatty acid side chains, since polyunsaturated fatty acids usually bind to sn-2 in vivo. This fact agrees with the fact that saturated fatty acids are usually bound to sn-1 and unsaturated fatty acids are bound to sn-2 in lipids present in vivo. The ratio of the ionic strength of the standard PC, which actually binds (18:0)/(18:1) and (16:0)/(18:1) as fatty acid side chains, to the ionic strength ratio of the biological sample. , the binding modes of sn-1 and sn-2 can be clearly seen. From the ionic strength ratio, it was proved that the saturated fatty acid was bound to sn-1 and the unsaturated fatty acid was bound to sn-2. Regarding PC (16:0/18:0), there is a difference in the ionic strength ratio between the standard and the biological sample, so the PC (16:0/18:0) of the biological sample does not necessarily have sn-1 of 16: It is not 0, and about half of them are thought to have 18:0 bound to sn-1. In this way, even if only a biological sample is used, quantification is performed without knowing which of sn-1 and sn-2 the fatty acid side chain is bound to. Using the ionic strength ratio, it is also possible to determine to which of sn-1 and sn-2 how much fatty acid is bound.

According to the present invention, a lipid profiling system, a lipid profiling method, and a lipid profiling program are provided that are capable of calculating quantitative ratios between different lipid molecules having two or more fatty acid side chains.

10... lipid profiling system, 20... tandem mass spectrometer, 110... acquisition unit, 120... lipid amount ratio calculation unit, 130... lipid concentration acquisition unit, 140... lipid concentration calculation unit, 150... output unit, 160... storage unit, HG: head group, L: lipid, CO: concentration, LR: lipid amount ratio, FC: fatty acid side chain, LC: lipid class, sn: stereospecifically numbered, LPC: lysophosphatidylcholine, LPE: lysophosphatidylethanol Amine

Claims (10)

A lipid system that performs lipid profiling of a sample containing multiple types of lipids based on the analysis results of a tandem mass spectrometer,
an acquiring unit that acquires, from the analysis results, the type of the fatty acid side chain of a lipid having two or more fatty acid side chains and the product ionic strength of the fatty acid side chain for each of the plurality of types of lipids belonging to the same lipid class; ,
Using the obtained product ionic strength, the product ionic strength of the first type of fatty acid side chain possessed by the first type of lipid and the second type of lipid belonging to the same lipid class as the first type of lipid possessed a lipid amount ratio calculation unit that calculates the amount ratio between the first type of lipid and the second type of lipid based on the ratio of the product ionic strength of the first type of fatty acid side chain;
A lipid profiling system comprising: The first type of lipid has the first type of fatty acid side chain and the second type of fatty acid side chain,
The lipid amount ratio calculation unit uses the acquired product ion intensity,
The product ionic strength of the second type of fatty acid side chain possessed by the first type of lipid and the product ionic strength of the second type of fatty acid side chain possessed by the third type of lipid belonging to the same lipid class as the first type of lipid. Calculate the quantitative ratio between the first type of lipid and the third type of lipid based on the ratio of the product ionic strength,
A lipid profiling system according to claim 1 . The second type of lipid has the first type of fatty acid side chain and the third type of fatty acid side chain,
The lipid amount ratio calculation unit further uses the acquired product ion intensity,
The product ionic strength of the third type of fatty acid side chain possessed by the second type of lipid and the product ionic strength of the third type of fatty acid side chain possessed by the fourth type of lipid belonging to the same lipid class as the first type of lipid. Calculate the quantitative ratio between the second type of lipid and the fourth type of lipid based on the ratio of the product ionic strength,
A lipid profiling system according to claim 2. The lipid amount ratio calculation unit further
Hypothetical first correction coefficient indicated by the ratio of the product ionic strength of the first type of fatty acid side chain and the product ionic strength of the second type of fatty acid side chain possessed by the first type of lipid to be the slope standard calibration curve and
Hypothetical second correction coefficient indicated by the ratio of the product ionic strength of the first type of fatty acid side chain and the product ionic strength of the third type of fatty acid side chain possessed by the second type of lipid is assumed to be the slope create a standard calibration curve and
A lipid profiling system according to claim 2. a concentration acquisition unit that acquires the concentration of the first type of lipid;
Based on the concentration of the first type of lipid acquired by the concentration acquisition unit and the quantitative ratio between the first type of lipid and the second type of lipid, the concentration of the second type of lipid is determined. A lipid concentration calculation unit to calculate,
The lipid profiling system of claim 1, further comprising: The first type of lipid has the first type of fatty acid side chain and the second type of fatty acid side chain,
The lipid amount ratio calculation unit uses the acquired product ion intensity,
The product ionic strength of the second type of fatty acid side chain possessed by the first type of lipid and the product ionic strength of the second type of fatty acid side chain possessed by the third type of lipid belonging to the same lipid class as the first type of lipid. Calculate the quantitative ratio between the first type of lipid and the third type of lipid based on the ratio of the product ionic strength,
The lipid concentration calculator,
of the third type of lipid based on the concentration of the first type of lipid obtained by the concentration obtaining unit and the quantitative ratio between the first type of lipid and the third type of lipid; calculate the concentration,
Lipid profiling system according to claim 5. The second type of lipid has the first type of fatty acid side chain and the third type of fatty acid side chain,
The lipid amount ratio calculation unit further uses the acquired product ion intensity,
The product ionic strength of the third type of fatty acid side chain possessed by the second type of lipid and the product ionic strength of the third type of fatty acid side chain possessed by the fourth type of lipid belonging to the same lipid class as the first type of lipid. Calculate the quantitative ratio between the second type of lipid and the fourth type of lipid based on the ratio of the product ionic strength,
The lipid concentration calculator,
Calculate the concentration of the fourth type of lipid based on the concentration of the second type of lipid and the quantitative ratio between the second type of lipid and the fourth type of lipid;
Lipid profiling system according to claim 5. said lipid class is selected from the group consisting of diacylglycerols, triacylglycerols, phosphatidylcholines, phosphatidylethanolamines, phosphatidic acids, phosphatidylserines, phosphatidylinositols, and phosphatidylglycerols A lipid profiling system according to any one of claims 1-7. analyzing a sample containing multiple types of lipids with a tandem mass spectrometer;
A lipid having two or more fatty acid side chains obtained by the analysis of the tandem mass spectrometer, the types of the fatty acid side chains, and the types of the fatty acid side chains of the plurality of types of lipids belonging to the same lipid class obtaining a product ionic strength for each type of lipid;
Using the obtained product ionic strength, the product ionic strength of the first type of fatty acid side chain possessed by the first type of lipid and the second type of lipid belonging to the same lipid class as the first type of lipid possessed calculating the quantitative ratio between the first type of lipid and the second type of lipid based on the ratio of the product ionic strength of the first type of fatty acid side chain;
A method of lipid profiling, comprising: to the computer,
From the analysis results of a sample containing multiple types of lipids analyzed by a tandem mass spectrometer, lipids having two or more fatty acid side chains and belonging to the same lipid class, the multiple types of lipids belong to the same lipid class. obtaining the type and product ionic strength of the fatty acid side chain for each type of the lipid;
Using the obtained product ionic strength, the product ionic strength of the first type of fatty acid side chain possessed by the first type of lipid and the second type of lipid belonging to the same lipid class as the first type of lipid possessed calculating the quantitative ratio between the first type of lipid and the second type of lipid based on the ratio of the product ionic strength of the first type of fatty acid side chain;
A lipid profiling program for running

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