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WO2018016645A1 - Nouveau phospholipide et ses utilisations ainsi que développement d'un procédé de mesure de séparation de phospholipides - Google Patents

Nouveau phospholipide et ses utilisations ainsi que développement d'un procédé de mesure de séparation de phospholipides Download PDF

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Publication number
WO2018016645A1
WO2018016645A1 PCT/JP2017/026563 JP2017026563W WO2018016645A1 WO 2018016645 A1 WO2018016645 A1 WO 2018016645A1 JP 2017026563 W JP2017026563 W JP 2017026563W WO 2018016645 A1 WO2018016645 A1 WO 2018016645A1
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Prior art keywords
lysophosphatidylinositol
phosphate
monophosphate
diphosphate
phosphatidylinositol
Prior art date
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English (en)
Japanese (ja)
Inventor
雄彦 佐々木
広樹 中西
将己 石川
紀子 上野
賢史 江口
純子 佐々木
中西　貴代
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Akita Lipid Technologies
Akita Lipid Technologies LLC
Akita University NUC
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Akita Lipid Technologies
Akita Lipid Technologies LLC
Akita University NUC
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Priority to JP2018528909A priority Critical patent/JP7549306B2/ja
Publication of WO2018016645A1 publication Critical patent/WO2018016645A1/fr
Anticipated expiration legal-status Critical
Priority to JP2022085341A priority patent/JP2022118008A/ja
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/117—Esters of phosphoric acids with cycloaliphatic alcohols
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G01N27/622—Ion mobility spectrometry
    • G01N27/623—Ion mobility spectrometry combined with mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to a novel phospholipid and use thereof. Furthermore, the present invention relates to a phospholipid separation measurement method. More specifically, the present invention relates to a technique for specifying the position of a phosphate group of phosphatidylicitol phosphate and / or lysophosphatidylinositol phosphate while keeping an acyl group intact. Specifically, the present invention provides a phospholipid separation measurement method using mass spectrometry (MS), optionally in combination with chromatography or ion mobility separation (IMS).
  • MS mass spectrometry
  • IMS ion mobility separation
  • Lipids are essential components of life as membrane constituents, energy sources, and signal transduction molecules.
  • phospholipids are an important group of molecules that regulates various cellular functions as spatiotemporal regulators that target intracellular proteins to cell membranes and organelles and control their activity at those locations.
  • it since it is sparingly soluble in water and not directly under the control of genes, it has limited analysis and has become a biochemical research field where research has not progressed.
  • the present inventors challenged such an undeveloped field by using a new technique of producing a genetically modified mouse having a phospholipid metabolizing enzyme or a phospholipid binding domain.
  • the research of the present inventors has become a pioneer in the world, and elucidation of the biological function regulation mechanism by intracellular phospholipid has been developed so that it can be regarded as a new research field.
  • various medical conditions such as various cancers, allergies, hepatic steatosis / fibrosis, cardiac dysfunction, neurodegeneration, enterocolitis, etc. were found one after another, and abnormal metabolic enzymes of individual phospholipids were found one after another.
  • Non-patent Document 1 Non-patent Document 1
  • Non-patent Document 1 lipid detection, identification and quantification methods have not been developed yet. Unlike genes, the type and number of lipids that make up living organisms are still uncertain, and many unknown lipids are thought to be sleeping without being discovered.
  • Phosphoinositide also referred to as phosphatidylinositol phosphate, phosphatidylinositol phosphate (salt), etc.
  • Phosphoinositide involved in many diseases includes 8 classes (phosphatidylinositol, phosphatidylinositol-3-monophosphate, phosphatidylinositol-4-monophosphate) Acid, phosphatidylinositol-5-monophosphate, phosphatidylinositol-3,4-diphosphate, phosphatidylinositol-3,5-diphosphate, phosphatidylinositol-4,5-diphosphate, phosphatidylinositol-3,4 , 5-triphosphate). These lipids or metabolites may
  • the present inventors have begun to develop a new lipid analysis method based on mass spectrometry.
  • the merit of using mass spectrometry is that it is possible to analyze biological samples (human clinical specimens, etc.) that could not be analyzed without using the radioisotope label to be analyzed.
  • the composition of the lysophospholipid according to the present invention can be found.
  • Established a new measurement method for the phosphatidylinositol phosphate group also referred to as “PIPs” in this specification, which is extremely difficult to measure due to the presence of a small amount in the living body and multivalent phosphorylation. .
  • LPIPs lysophosphatidylinositol phosphate group
  • phosphoinositide and lysophosphoinositide also referred to as lysophosphatidylinositol phosphate, lysophosphatidylinositol phosphate (salt), etc.
  • lysophosphatidylinositol phosphate lysophosphatidylinositol phosphate (salt), etc.
  • acyl group is obtained by chromatography-mass spectrometry.
  • the present invention relates to all 8 classes of phosphoinositides and all 8 classes of lysophosphoinositides with information on acyl groups (fatty acid side chains) by liquid column chromatography / mass spectrometry (LC-MS) or ion mobility separation.
  • LC-MS liquid column chromatography / mass spectrometry
  • IMS-MS mass spectrometry
  • the present invention provides the following.
  • (Item 1A) A compound which is lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate or lysophosphatidylinositol triphosphate.
  • (Item 2A) 1-acyl-lysophosphatidylinositol monophosphate, 2-acyl-lysophosphatidylinositol monophosphate, 1-acyl-lysophosphatidylinositol diphosphate, 2-acyl-lysophosphatidylinositol diphosphate, 1-acyl-lysophosphatidyl The compound according to item 1A, which is inositol triphosphate or 2-acyl-lysophosphatidylinositol triphosphate.
  • (Item 3A) 1-acyl-lysophosphatidylinositol-3-monophosphate, 1-acyl-lysophosphatidylinositol-4-monophosphate, 1-acyl-lysophosphatidylinositol-5-monophosphate, 2-acyl-lysophosphatidylinositol- 3-monophosphate, 2-acyl-lysophosphatidylinositol-4-monophosphate, 2-acyl-lysophosphatidylinositol-5-monophosphate, 1-acyl-lysophosphatidylinositol-3.4-diphosphate, 1-acyl-lysophosphatidylinositol-3.5-diphosphate, 1-acyl-lysophosphatidylinositol-4.5-diphosphate, 2-acyl-lysophosphatidylinositol-3,4-diphosphate, 2- Acy
  • Item 4A Item 1A-3A which is 2-acyl-lysophosphatidylinositol monophosphate, 2-acyl-lysophosphatidylinositol diphosphate, 1-acyl-lysophosphatidylinositol triphosphate or 2-acyl-lysophosphatidylinositol triphosphate
  • 2-acyl-lysophosphatidylinositol monophosphate 2-acyl-lysophosphatidylinositol diphosphate
  • 1-acyl-lysophosphatidylinositol triphosphate 2-acyl-lysophosphatidylinositol triphosphate
  • 2-acyl-lysophosphatidylinositol triphosphate 2-acyl-lysophosphatidylinositol triphosphate
  • (Item 6A) A composition comprising the compound according to any one of items 1A to 5A for use as a disease marker.
  • (Item 7A) Item 6.
  • (Item 8A) The composition of item 7A, wherein the cancer comprises prostate cancer.
  • (Item 9A) Item 6.
  • the method for producing a compound according to any one of Items 1A to 5A comprising a step of deacylating phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate by contacting with alkylamine. .
  • (Item 10A) Item 9.
  • the production method according to Item 9A wherein the alkylamine is methylamine.
  • Item 11A Item 10. The production method according to Item 9A or Item 10A, wherein the deacylation is a milder condition than a condition for dediacylation.
  • the method according to Item 11A wherein the mild condition is achieved by shortening a reaction time, reducing a concentration of the alkylamine, a reaction temperature, or a combination thereof among the conditions for dediacylation.
  • the mild condition is that methylamine is used as the alkylamine, the reaction time is 10 minutes or less, or the concentration of the methylamine is about 11% or less, the reaction temperature is 53 ° C.
  • (Item 14A) (A) a step of providing phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate having different mass acyl groups at the 1-position and the 2-position, wherein the acyl group having a desired mass is Existing in a desired position selected from the 1st or 2nd position; (B) deacylating the phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate by contacting with an alkylamine, and (C) an acyl having a desired mass, if necessary.
  • lysophosphatidylinositol monophosphate Removing lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate or lysophosphatidylinositol triphosphate having a group, lysophosphatidylinositol monophosphate having a desired acyl group at a desired position, lysophosphatidylinositol
  • (Item 15A) Concentrating the phospholipid by contacting the sample with an anion exchange resin; Protecting a phosphate group in the acidic phospholipid with a protecting group; and detecting, identifying or quantifying lysophosphatidylinositol phosphate in the phospholipid by mass spectrometry; For detecting, identifying or quantifying lysophosphatidylinositol phosphate.
  • (Item 16A) The method according to Item 15A, further comprising detecting, identifying or quantifying phosphatidylinositol phosphate in the phospholipid.
  • (Item 17A) The method of item 15A or 16A, wherein the protecting group comprises an alkyl group.
  • (Item 18A) The method according to any one of Items 15A to 17A, wherein the protecting group comprises a methyl group.
  • (Item 19A) The method according to any one of Items 15A to 18A, wherein the mass spectrometry includes a selective reaction monitoring method (SRM) using a triple quadrupole mass spectrometer.
  • SRM selective reaction monitoring method
  • (Item 20A) Further, any one of items 15A to 19A, further comprising a step of detecting, identifying or quantifying the fatty acid side chain and / or phosphate group of the lysophosphatidylinositol phosphate using liquid column chromatography or ion mobility separation. 2. The method according to item 1.
  • (Item 21A) Item 20.
  • (Item 24A) A method for measuring, detecting or identifying phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate in a sample, the method comprising: A) applying the sample to mass spectrometry (MS); and B) a peak in the MS And locating the phosphate group of the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate according to the elution position of the phosphatidylinositol phosphate.
  • the method according to Item 24A further comprising applying the sample to chromatography in the step A).
  • the mass spectrometry is liquid column chromatography-mass spectrometry (LC-MS);
  • Item 27A Item 26. The method according to any one of Items 24A to 26A, wherein the total molecular weight of the acyl groups contained in the phosphatidylinositol phosphate is also specified in the specifying step.
  • (Item 28A) The method according to any one of Items 24A to 27A, wherein in the identifying step, the kind of acyl group contained in the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate is also identified.
  • (Item 29A) The method according to any one of items 24A to 28A, wherein the position of the phosphate group of said phosphatidylinositol phosphate and lysophosphatidylinositol phosphate is specified in the same run.
  • the condition includes not performing a deacylation treatment.
  • (Item 33A) The method according to any one of items 26A to 32A, wherein the liquid column chromatography is selected from the group consisting of a chiral column, a reverse phase column, and a column chromatography that separates a hydrophilic group and a hydrophobic group.
  • (Item 34A) The method of any one of items 24A-33A, wherein the liquid column chromatography is combined with ion mobility separation.
  • Item 35A The method of any one of items 24A-34A, further comprising quantifying the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate.
  • (Item 36A) The method of any one of items 24A-35A, wherein the sample is an intact sample.
  • (Item 37A) The method according to any one of Items 24A to 36A, wherein the identification is performed without labeling the sample.
  • (Item 38A) The mass spectrometry is ion mobility separation-mass spectrometry (IMS-MS);
  • the specifying step B) is a step of specifying the position of the phosphate group of the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate by the elution position of the peak in the IMS-MS.
  • (Item 39A) The method of item 38A, comprising any one or more of the items 25A-33A, 35A-37A.
  • (Item 40A) An apparatus for measuring, detecting or identifying the position of acyl group and phosphate group of phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate in a sample under conditions where mass spectrometry (MS) and phosphatidylinositol phosphate are not decomposed And an application unit that applies the sample to the MS.
  • MS mass spectrometry
  • phosphatidylinositol phosphate an application unit that applies the sample to the MS.
  • the device according to Item 40A further comprising a chromatography device.
  • Item 42A Item 40A, wherein the mass spectrometry is liquid column chromatography-mass spectrometry (LC-MS), and the application unit is an application unit that applies the sample to the LC-MS under conditions in which phosphatidylinositol phosphate is not decomposed. Or the apparatus of 41A.
  • Item 43A 42.
  • the apparatus according to any one of items 40A-43A, further comprising means for detecting or quantifying said phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate.
  • the mass spectrometry is ion mobility separation-mass spectrometry (IMS-MS), and the application section is an application section that applies the sample to the IMS-MS under the condition that phosphatidylinositol phosphate is not decomposed.
  • IMS-MS ion mobility separation-mass spectrometry
  • (Item 46A) A method for separating or purifying phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate in a sample according to the position of a phosphate group while retaining an acyl group, the method comprising: A) Steps applied to graphy-mass spectrometry (LC-MS) or ion mobility separation-mass spectrometry (IMS-MS); and B) the elution position of the peak from the eluate of LC or IMS in the LC-MS or IMS-MS Collecting phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate having a phosphate group at the position of interest specified by.
  • LC-MS graphy-mass spectrometry
  • IMS-MS ion mobility separation-mass spectrometry
  • (Item 48A) 47 The method according to Item 47A, wherein the number of the phosphatidylinositol phosphate and / or the lysophosphatidylinositol phosphate contained in the mixture is less than the number of types of the phosphate group.
  • the present invention provides the following.
  • (1B) a method for measuring, detecting or identifying phosphatidylinositol phosphate in a sample comprising: A) applying the sample to liquid column chromatography-mass spectrometry (LC-MS); and B) A method comprising the step of locating the phosphate group of the phosphatidylinositol phosphate by the elution position of the peak in LC-MS.
  • LC-MS liquid column chromatography-mass spectrometry
  • IMS-MS ion mobility separation-mass spectrometry
  • IMS-MS ion mobility separation-mass spectrometry
  • a method for separating or purifying phosphatidylinositol phosphate in a sample according to the position of a phosphate group while retaining an acyl group comprising: A) liquid column chromatography-mass spectrometry of the sample (LC-MS) or a step applied to ion mobility separation-mass spectrometry (IMS-MS); and B) from the eluate of the LC-MS or IMS-MS to the target position specified by the elution position of the peak Collecting a phosphatidylinositol phosphate having a phosphate group.
  • LC-MS liquid column chromatography-mass spectrometry of the sample
  • IMS-MS ion mobility separation-mass spectrometry
  • a phosphatidyl group having a phosphate group at a kind of position less than the number of kinds of the phosphate group of the phosphatidylinositol phosphate included in the mixture from a mixture of two or more phosphate groups of the phosphatidylinositol phosphate A method for producing a sample comprising inositol phosphate, comprising: A) applying the sample to liquid column chromatography-mass spectrometry (LC-MS) or ion mobility separation-mass spectrometry (IMS-MS); and B) from the eluate of the LC-MS or IMS-MS, Collecting a fraction containing a phosphatidylinositol phosphate having a phosphate group at a target position specified by an elution position of a peak.
  • LC-MS liquid column chromatography-mass spectrometry
  • IMS-MS ion mobility separation-mass spectrometry
  • the present invention relates to an analytical method that can separate and measure eight PIPs and LPIPs isomers intact using a chiral column.
  • An exemplary analysis uses MRM / SRM with triple quadrole mass spectrometer. Q1 sets an intact precursor ion, and Q3 sets DAG or MAG from which inositol phosphate is eliminated as a product ion.
  • the present invention also provides the following.
  • (1C) A compound which is lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate or lysophosphatidylinositol triphosphate.
  • (2C) 1-acyl-lysophosphatidylinositol monophosphate, 2-acyl-lysophosphatidylinositol monophosphate, 1-acyl-lysophosphatidylinositol diphosphate, 2-acyl-lysophosphatidylinositol diphosphate, 1-acyl
  • the compound according to item 1C which is lysophosphatidylinositol triphosphate or 2-acyl-lysophosphatidylinositol triphosphate.
  • (3C) 1-acyl-lysophosphatidylinositol-3-monophosphate, 1-acyl-lysophosphatidylinositol-4-monophosphate, 1-acyl-lysophosphatidylinositol-5-monophosphate, 2-acyl-lyso Phosphatidylinositol-3-monophosphate, 2-acyl-lysophosphatidylinositol-4-monophosphate, 2-acyl-lysophosphatidylinositol-5-monophosphate, 1-acyl-lysophosphatidylinositol-3-4-2 Phosphoric acid, 1-acyl-lysophosphatidylinositol-3.5-diphosphate, 1-acyl-lysophosphatidylinositol-4.5-diphosphate, 2-acyl-lysophosphatidylinositol-3,4-diphosphate 2-acyl
  • Item 1C which is 2-acyl-lysophosphatidylinositol monophosphate, 2-acyl-lysophosphatidylinositol diphosphate, 1-acyl-lysophosphatidylinositol triphosphate or 2-acyl-lysophosphatidylinositol triphosphate
  • 2-acyl-lysophosphatidylinositol monophosphate 2-acyl-lysophosphatidylinositol diphosphate
  • 1-acyl-lysophosphatidylinositol triphosphate 2-acyl-lysophosphatidylinositol triphosphate
  • (13C) A step of providing phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate having acyl groups with different masses at the 1-position and the 2-position, and having a desired mass An acyl group is present at a desired position selected from the 1-position or 2-position; (B) contacting the phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate with an alkylamine; And (C) optionally removing lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate or lysophosphatidylinositol triphosphate having a desired mass of acyl group.
  • a method for detecting, identifying or quantifying lysophosphatidylinositol phosphate comprising the step of detecting, identifying or quantifying lysophosphatidylinositol phosphate in said phospholipid by an analytical method.
  • the protecting group comprises an alkyl group.
  • the protecting group comprises a methyl group.
  • the mass spectrometry includes a selective reaction monitoring method (SRM) using a triple quadrupole mass spectrometer.
  • the present invention provides new lipids to elucidate the vital functions of these lipids and can be applied to medical applications.
  • new drug discovery targets can be presented.
  • the present invention can provide a new diagnosis and detection method by providing a new measurement method.
  • phosphoinositide and / or lysophosphoinositide can be separated at the position of a phosphate group and measured, detected or identified in an intact state. Analysis can be performed. Such information is useful in biological information, pharmacological information, drug development, treatment, diagnosis, and the like.
  • FIG. 1A is a schematic diagram for explaining the principle of “measurement and detection method by SRM in a triple quadrupole mass spectrometer”.
  • FIG. 1B shows the experimental results for studying the conditions for the synthesis of the compounds of the present invention.
  • LPIP1 is not produced under the conventional deacylation reaction conditions, but LPIP1 can be obtained by shortening the reaction time (upper stage) or decreasing the methylamine concentration (lower stage).
  • the vertical axis represents the yield (%) of the reaction product, and the horizontal axis represents time (minutes).
  • FIG. 1C shows the results of examining the reaction temperature. 17: 0/20: 4 PI4P was used as a raw material.
  • the products are 17: 0 LPIP1 and 20: 4 LPIP1.
  • FIG. 2A shows the identification data of a synthetic product of the compound of the present invention.
  • FIG. 2B shows the identification data of a synthetic product of the compound of the present invention.
  • FIG. 2C shows identification data for a synthetic product of the compounds of the present invention.
  • FIG. 2D shows identification data for a synthetic product of the compounds of the present invention.
  • FIG. 2E shows identification data for a synthetic product of the compounds of the present invention.
  • FIG. 2F shows identification data of a synthetic product of the compound of the present invention.
  • FIG. 2G shows identification data for a synthetic product of the compounds of the invention.
  • FIG. 3A shows two types of LPI produced by a mild deacylation reaction (see FIG. 1) of 37: 4 PI (4) P (sn-1 17: 0, sn-2 20: 4 PI (4) P). (4) About P, the result of having performed the exact mass spectrometry of the fragment derived from LPI (4) P and LPI (4) P is shown. The vertical axis represents relative abundance, and the horizontal axis represents time (minutes).
  • FIG. 3B shows the structural formula of 37: 4 PI (4) P, the product after the deacylation reaction, and fragments thereof.
  • 3C shows two types of LPIs produced by a mild deacylation reaction of 37: 4 PI (4,5) P2 (sn-1 17: 0, sn-2 20: 4 PI (4,5) P2).
  • 4,5) P2 shows the results of accurate mass analysis of LPI (4,5) P2 and fragments derived from LPI (4,5) P2.
  • the vertical axis represents relative abundance, and the horizontal axis represents time (minutes).
  • FIG. 3D shows the structural formula of 37: 4 PI (4,5) P2, the product after the deacylation reaction, and its fragment.
  • FIG. 3E shows a two-dimensional reaction resulting from a mild deacylation reaction of 37: 4 PI (3,4,5) P3 (sn-1 17: 0, sn-2 20: 4 PI (3,4,5) P3).
  • FIG. 4A represents an overview of the spin system of inositol phospholipids. A portion surrounded by a solid line indicates a group of 1 H nuclei connected by continuous 3 J coupling, and a portion surrounded by a broken line indicates a portion where 1 H nuclei and 31 P nuclei are 3 J coupled.
  • FIG. 4A represents an overview of the spin system of inositol phospholipids. A portion surrounded by a solid line indicates a group of 1 H nuclei connected by continuous 3 J coupling, and a portion surrounded by a broken line indicates a portion where 1 H nuclei and 31 P nuclei are 3 J coupled.
  • FIG. 4C shows the chemical structure observed from the 1 H-1D, TOCSY spectrum of Sample 1 of Example 1 (16: 0 LPI4P).
  • the chemical structure observed from the structural formula of 16: 0 LPI4P and 1 H-1D and TOCSY spectra is shown.
  • Fatty acid and glycerol skeleton C1 and C3 signals were observed.
  • a signal considered to be an inositol ring was weakly observed. No signal of polyunsaturated fatty acids was observed.
  • FIG. 4D shows the chemical structure observed from the 1 H-1D, TOCSY spectrum of Sample 2 of Example 1 (17: 0 LPI (4,5) P2).
  • FIG. 4E shows the chemical structure observed from the 1 H-1D, TOCSY spectrum of sample 3 of Example 1 (18: 0 LPI (4,5) P2).
  • 18: 0 shows the chemical structure observed from the structural formula of LPI (4,5) P2 and the signals of 1 H-1D and TOCSY spectra.
  • FIG. 4F shows the chemical structure observed from the 1 H-1D, TOCSY spectrum of Sample 5 of Example 1 (17: 0/20: 4 PI (4,5) P2). 17: 0/20: 4 This shows the chemical structure observed from the structural formula of PI (4,5) P2 and the signals of 1 H-1D and TOCSY spectra. Fatty acid and glycerol skeleton C1, C2, and C3 signals were observed.
  • FIG. 5A-5D show data relating to the presence of a compound of the invention in a biological sample (cultured cancer cell line).
  • FIG. 5A shows the presence of LPIPs in HEK293T cells.
  • the vertical axis represents the abundance (pmol) of the compound relative to 1 nmol of phosphatidylserine (PS), and the horizontal axis represents the molecular species.
  • FIG. 5B shows the presence of LPIPs in Jurkat cells.
  • the vertical axis represents the abundance (pmol) of the compound relative to 1 nmol of phosphatidylserine (PS), and the horizontal axis represents the molecular species.
  • FIG. 5C shows the results of accurate mass measurement of LPIP3 and fragments thereof present in HEK293T cells. The vertical axis represents relative abundance, and the horizontal axis represents m / z (mass / number of charges).
  • FIG. 5D shows the coincidence of elution times of LPIP3 present in HEK293T cells and fragments thereof. The vertical axis represents relative abundance, and the horizontal axis represents time (minutes). 6A-6D show data relating to the presence in biological samples (mouse, human) for the compounds of the present invention.
  • FIG. 6A shows that LPIPs are present in the mouse heart, large intestine, liver, brain (whole brain), and spleen.
  • the vertical axis represents the abundance (pmol) of the compound relative to 1 nmol of phosphatidylserine (PS), and the horizontal axis represents the cell type.
  • FIG. 6B shows the presence of LPIPs in mouse serum.
  • the vertical axis represents the abundance (fmol) of the compound relative to 1 ⁇ L of serum, and the horizontal axis represents the molecular species.
  • FIG. 6C shows the presence of LPIPs in mouse plasma.
  • the vertical axis represents the abundance (fmol) of the compound relative to 1 ⁇ L of plasma, and the horizontal axis represents the molecular species.
  • FIG. 6D shows the presence of LPIPs in human serum.
  • the vertical axis represents the abundance (fmol) of the compound relative to 1 ⁇ L of serum, and the horizontal axis represents the molecular species.
  • FIG. 7A shows mouse prostate weight in the upper row (vertical axis indicates weight (mg), horizontal axis indicates tissue type), and lower row shows prostate HE-stained image. Indicates that cancer develops.
  • FIG. 7B shows that LPIPs levels in the prostate increase with the development of prostate cancer.
  • the vertical axis represents LPIPs level (AU (arbitrary unit)) for 1 nmol of phosphatidylserine (PS), the horizontal axis Ctrl indicates normal mice, and PTEN KO expresses the tumor suppressor gene Pten in a prostate-specific manner.
  • FIG. 8 shows that LPIP3 is generated in myelocytes upon stimulation with complement component 5a, which is a inflammatory substance.
  • the vertical axis indicates the abundance (pmol) of LPIP3 with respect to 10 6 cells, the horizontal axis “ ⁇ ” indicates that stimulation by the complement component 5a is not performed, and “+” indicates the complement component This means that stimulation by 5a is being performed.
  • FIGS. 9A and 9B are diagrams showing the separation of 17: 0/20: 4 phosphoinositides on a chiral column.
  • Each graph is a chromatogram when the phosphoric acid group phosphoinositide sample displayed is measured with a mass spectrometer, the horizontal axis represents retention time (minutes), and the vertical axis represents peak-top signal intensity (cps). Represents the relative signal intensity with respect to 100%.
  • the left column of FIG. 9A shows PI
  • the center column shows PI (3) P
  • the right column shows their mixture (PIP1mix).
  • the second column from the right shows PI (3,5) P, PI (3,4) P2PI (4,5) P2 and mixtures thereof, respectively.
  • the left column of FIG. 9B shows PI (3,5) P2, PI (3,4) P2 and PI (4,5) P2 from the top, the center shows their mixture (PIP2mix), and the right column shows PI (3,4,5) P3 is shown.
  • 9A and 9B are diagrams showing the separation of 17: 0/20: 4 phosphoinositides on a chiral column. Each graph is a chromatogram when the phosphoric acid group phosphoinositide sample displayed is measured with a mass spectrometer, the horizontal axis represents retention time (minutes), and the vertical axis represents peak-top signal intensity (cps).
  • FIG. 9A Represents the relative signal intensity with respect to 100%.
  • the left column of FIG. 9A shows PI
  • the center column shows PI (3) P, PI (4) P and PI (5) P from the top, respectively, and the right column shows their mixture (PIP1mix).
  • the second column from the right shows PI (3,5) P, PI (3,4) P2PI (4,5) P2 and mixtures thereof, respectively.
  • the left column of FIG. 9B shows PI (3,5) P2, PI (3,4) P2 and PI (4,5) P2 from the top, the center shows their mixture (PIP2mix), and the right column shows PI (3,4,5) P3 is shown.
  • FIG. 9A shows PI
  • the center column shows PI (3) P, PI (4) P and PI (5) P from the top, respectively
  • the right column shows their mixture (PIP1mix).
  • the second column from the right shows PI (3,5) P, PI (3,4) P2PI (4,5) P2 and mixtures thereof,
  • 10 shows seven different amounts (0.02, 0.05, 0.1, 0.2, 5, 10, 50 pmol) of the indicated phosphate group 17: 0/20: 4 phosphoinositide.
  • the calibration curve prepared from the signal intensity (peak area) when measured with a mass spectrometer (repeatedly measured four times for each injection amount) is shown.
  • the average of 4 repeated measurements is represented by black dots, and the standard deviation is represented by error bars.
  • the upper row shows PI, PI (3) P, and PI (4) P from the left
  • the middle row shows PI (5) P, PI (3,5) P, and PI (3,4) P2 from the left
  • the lower row From the left, PI (4,5) P2, PI (3,4,5), P3 are shown.
  • FIG. 11 shows fluctuations in signal intensity (cps) when 10 pmol of 17: 0/20: 4 phosphoinositide of the displayed phosphate group was measured 50 times with a mass spectrometer.
  • the vertical axis represents signal intensity (cps), and the horizontal axis represents the number of injections.
  • PI (3) P gives the first strongest signal strength
  • PI (4) P gives the second strongest signal strength
  • PI (5) P gives the third strongest signal strength.
  • PI (3,4) P2 gives the fourth strongest signal strength
  • PI (3,5) P2 gives the fifth strongest signal strength
  • PI gives the sixth strongest signal strength
  • PI (4, 5) P2 gave the seventh strongest signal intensity
  • PI (3,4,5) P3 gave the weakest signal intensity.
  • FIG. 12 shows the separation of 17: 0/20: 4 phosphoinositides (PI (3,4) P2, PI (3,5) P2, PI (4,5) P2) by ion mobility.
  • the horizontal axis represents compensation voltage (COV, V), and the vertical axis represents signal intensity (cps).
  • COV, V compensation voltage
  • cps signal intensity
  • FIG. 13 shows the change in phosphoinositide composition of cells when the cells were treated with H 2 O 2 as a result of measurement using a combination of a chiral column and a mass spectrometer.
  • the horizontal axis represents the retention time
  • the vertical axis represents the relative signal intensity when the peak top signal intensity (cps) is 100%.
  • FIGS. 14A to 14D show changes in the phosphoinositide composition for each diacylglycerol of the cells and the total amount of each phosphoinositide when the cells were treated with H 2 O 2 and quantification calculated from the measurement results of the chiral column-mass spectrometer Shown as a value.
  • the horizontal axis represents the type of diacylglycerol in phosphoinositide Tide, the amount of each phosphoinositide tide vertical axis contained in the cell 106 which is calculated as the average of four measurements a (pmol) Expressed with standard deviation.
  • pmol the average of four measurements a
  • the vertical axis represents the total amount of phosphoinositide Tide with the same phosphate substitutions contained in the cell 106 which is calculated as the average of four measurements a (pmol) with standard deviation
  • the horizontal axis represents the left To H 2 O 2 treatment at 0 min, 2 min, 5 min, and 15 min. It is a result of four repeated measurements, and the average of repeated measurements is shown together with a standard deviation (error bar).
  • ANOVA when statistical processing using the multiple comparison test of Tukey, that *** is p ⁇ 0.001, that ** is p ⁇ 0.01, *, p ⁇ Each represents 0.05.
  • 14A to 14D show changes in the phosphoinositide composition for each diacylglycerol of the cells and the total amount of each phosphoinositide when the cells were treated with H 2 O 2 and quantification calculated from the measurement results of the chiral column-mass spectrometer Shown as a value.
  • the horizontal axis represents the type of diacylglycerol in phosphoinositide Tide, the amount of each phosphoinositide tide vertical axis contained in the cell 106 which is calculated as the average of four measurements a (pmol) Expressed with standard deviation.
  • the results at the 0 minute point, the 2 minute point, the 5 minute point, and the 15 minute point of H 2 O 2 treatment are shown from the left.
  • the vertical axis represents the total amount of phosphoinositide Tide with the same phosphate substitutions contained in the cell 106 which is calculated as the average of four measurements a (pmol) with standard deviation
  • the horizontal axis represents the left To H 2 O 2 treatment at 0 min, 2 min, 5 min, and 15 min. It is a result of four repeated measurements, and the average of repeated measurements is shown together with a standard deviation (error bar).
  • FIGS. 14A to 14D show changes in the phosphoinositide composition for each diacylglycerol of the cells and the total amount of each phosphoinositide when the cells were treated with H 2 O 2 and quantification calculated from the measurement results of the chiral column-mass spectrometer Shown as a value.
  • the horizontal axis represents the type of diacylglycerol in phosphoinositide Tide, the amount of each phosphoinositide tide vertical axis contained in the cell 106 which is calculated as the average of four measurements a (pmol) Expressed with standard deviation.
  • pmol the average of four measurements a
  • the vertical axis represents the total amount of phosphoinositide Tide with the same phosphate substitutions contained in the cell 106 which is calculated as the average of four measurements a (pmol) with standard deviation
  • the horizontal axis represents the left To H 2 O 2 treatment at 0 min, 2 min, 5 min, and 15 min. It is a result of four repeated measurements, and the average of repeated measurements is shown together with a standard deviation (error bar).
  • ANOVA when statistical processing using the multiple comparison test of Tukey, that *** is p ⁇ 0.001, that ** is p ⁇ 0.01, *, p ⁇ Each represents 0.05.
  • 14A to 14D show changes in the phosphoinositide composition for each diacylglycerol of the cells and the total amount of each phosphoinositide when the cells were treated with H 2 O 2 and quantification calculated from the measurement results of the chiral column-mass spectrometer Shown as a value.
  • the horizontal axis represents the type of diacylglycerol in phosphoinositide Tide, the amount of each phosphoinositide tide vertical axis contained in the cell 106 which is calculated as the average of four measurements a (pmol) Expressed with standard deviation.
  • the results at the 0 minute point, the 2 minute point, the 5 minute point, and the 15 minute point of H 2 O 2 treatment are shown from the left.
  • the vertical axis represents the total amount of phosphoinositide Tide with the same phosphate substitutions contained in the cell 106 which is calculated as the average of four measurements a (pmol) with standard deviation
  • the horizontal axis represents the left To H 2 O 2 treatment at 0 min, 2 min, 5 min, and 15 min. It is a result of four repeated measurements, and the average of repeated measurements is shown together with a standard deviation (error bar).
  • FIG. 15 shows changes in the phosphoinositide composition in the thyroid gland (organ) of the mouse when the mouse is genetically modified as a result of measurement using a combination of a chiral column and a mass spectrometer.
  • the horizontal axis represents the retention time
  • the vertical axis represents the relative signal intensity when the peak top signal intensity (cps) is 100%.
  • FIGS. 16A to 16C show the change in phosphoinositide composition for each diacylglycerol and the total amount of each phosphoinositide in the thyroid gland (organ) of the mouse when the mouse is genetically modified from the measurement results of the chiral column-mass spectrometer. It is shown as a quantitative value. In the graphs of FIGS.
  • the horizontal axis represents the type of diacylglycerol in phosphoinositide
  • the vertical axis represents 3 times (wild type), 4 times (INPP4B ( ⁇ / ⁇ ), PTEN (+/ ⁇ ), And the amount (pmol) of each phosphoinositide contained in 1 mg of tissue calculated as an average of the INPP4B ( ⁇ / ⁇ ) PTEN (+/ ⁇ )) measurement, together with the standard deviation.
  • the results for wild type, INPP4B ( ⁇ / ⁇ ), PTEN (+/ ⁇ ), and INPP4B ( ⁇ / ⁇ ) PTEN (+/ ⁇ ) mice are shown from the left.
  • 16A to 16C show the change in phosphoinositide composition for each diacylglycerol and the total amount of each phosphoinositide in the thyroid gland (organ) of the mouse when the mouse is genetically modified from the measurement results of the chiral column-mass spectrometer. It is shown as a quantitative value. In the graphs of FIGS.
  • the horizontal axis represents the type of diacylglycerol in phosphoinositide
  • the vertical axis represents 3 times (wild type), 4 times (INPP4B ( ⁇ / ⁇ ), PTEN (+/ ⁇ ), And the amount (pmol) of each phosphoinositide contained in 1 mg of tissue calculated as an average of the INPP4B ( ⁇ / ⁇ ) PTEN (+/ ⁇ )) measurement, together with the standard deviation.
  • the results for wild type, INPP4B ( ⁇ / ⁇ ), PTEN (+/ ⁇ ), and INPP4B ( ⁇ / ⁇ ) PTEN (+/ ⁇ ) mice are shown from the left.
  • 16A to 16C show changes in the phosphoinositide composition for each diacylglycerol and the total amount of each phosphoinositide in the thyroid gland (organ) of the mouse when the mouse is genetically modified from the measurement results of the chiral column-mass spectrometer. It is shown as a quantitative value.
  • the vertical axis represents the average of three times (wild type), four times (INPP4B ( ⁇ / ⁇ ), PTEN (+/ ⁇ ), and INPP4B ( ⁇ / ⁇ ) PTEN (+/ ⁇ )) measurements.
  • the total amount (pmol) of phosphoinositides having the same phosphate substitution contained in 1 mg of tissue calculated as follows is expressed with standard deviation, and the horizontal axis from the left is wild type, INPP4B ( ⁇ / ⁇ ), PTEN (+/ ⁇ ) And INPP4B ( ⁇ / ⁇ ) PTEN (+/ ⁇ ) mice. 3 (wild type), 4 times (INPP4B ( ⁇ / ⁇ ), PTEN (+/ ⁇ ), and INPP4B ( ⁇ / ⁇ ) PTEN (+/ ⁇ )) repeated measurement results, the average of repeated measurements , With standard deviation (error bar).
  • FIG. 17 is a diagram showing that it is possible to separate phosphoric acid position isomers of lysophospholipid by column chromatography.
  • the horizontal axis represents the retention time
  • the vertical axis represents the relative signal intensity when the peak top signal intensity (cps) is 100%.
  • the left column shows the results of lyso PI (3) P, lyso PI (4) P, and lyso PI (5) P at 17: 0 from the top.
  • the right column shows the results of lyso PI (3, 5) P2, lyso PI (3,4) P2, and lyso PI (4, 5) P2 at 17: 0 from the top.
  • the present invention provides a novel phospholipid and a technology for measuring, detecting or identifying phospholipids phosphoinositide and lysophosphoinositide separated by subclass.
  • phosphatidylinositol phosphate or “phosphatidylinositol phosphate” are used interchangeably and refer to the sn-1 position and sn ⁇ of glycerol (also referred to as propane-1,2,3-triol, glycerol).
  • An inositol bonded to the sn-3 position is phosphorylated with X inositol bonded to the 2-position.
  • One, two or three phosphate groups can be attached, in this case referred to as “phosphatidylinositol X phosphate” (where X is one, two or three).
  • phosphatidylinositol X phosphates are collectively referred to as “phosphoinositide”, “phosphatidylinositol phosphates”, or “PIPs”.
  • the phosphate group can be indicated at a position on inositol, such as phosphatidylinositol-3-monophosphate, phosphatidylinositol-4-monophosphate, phosphatidylinositol-5-monophosphate, phosphatidylinositol-3,4 -Described as diphosphate, phosphatidylinositol-3,5-diphosphate, phosphatidylinositol-4,5-diphosphate, phosphatidylinositol-3,4,5-triphosphate, etc.
  • the present invention For the first time in the present invention, a method for identifying the position of the phosphate group of the PIPs while retaining the acyl group was provided.
  • the detailed information of the phosphate group of PIPs in the living body can be understood by using the identification method provided by the present invention.
  • the present invention is a detailed information that cannot be analyzed by the prior art in the sense that information in the living body can be analyzed in a true sense.
  • inositol is myo-inositol and the fatty acid bond type is an ester type.
  • PIPs with arbitrary acyl and phosphate group positions can be prepared, see, for example, Stuart J. et al. Conway et al. , Org. Biomol. Chem. , 2010, 8, 66-76, any kind of PIP can be manufactured. So far, phosphoinositides have been analyzed by separation of RI-label or non-label deacylated glycerophosphoinositides, or by using molecular probes and antibodies with specific binding proteins. In that sense, there are still no analytical methods that can measure intact phosphoinositide.
  • lysophosphatidylinositol phosphate or “lysophosphatidylinositol phosphate” is used interchangeably, and one acyl group is removed from phosphatidylinositol and inositol bonded to the sn-3 position is X Individual phosphorylated.
  • One, two or three phosphate groups can be attached, in this case referred to as “lysophosphatidylinositol X phosphate” (where X is one, two or three).
  • lysophosphatidylinositol X phosphates are collectively referred to as “lysophosphoinositide”, “lysophosphatidylinositol phosphates”, or “LPIPs”.
  • the phosphate group can be represented by a position on inositol, and is expressed as, for example, lysophosphatidylinositol-3,4,5-triphosphate.
  • LPIPs are also present in the living body by using the identification method provided by the present invention.
  • the position of the phosphate group could be identified for LPIPs.
  • inositol is myo-inositol and the fatty acid bond type is an ester type.
  • sn- is an abbreviation for “Stereospecifically Numbered”, and is used when the carbon atoms of a glycerin derivative are represented by stereospecific numbering. If the glycerin derivative is racemic, add rac-; if the stereochemistry is unknown, add X-.
  • LPIPs There are two types of LPIPs, sn-1-LPIPs or sn-2-LPIPs, depending on the position of the acyl group binding. For the actual display, the position of the acyl group is used as it is, such as 1-acyl-phosphatidylinositol X phosphate, 2-acyl-phosphatidylinositol X phosphate.
  • the corresponding PIPs can be produced using this and applied to the synthesis method of the present invention to produce LPIPs having the desired fatty acid group.
  • Specific embodiments of the present invention include the following. Monophosphate, sn-1-position 1-butanyl-lysophosphatidylinositol 4-monophosphate; 1-hexanyl-lysophosphatidylinositol 4-monophosphate; 1-octanyl-lysophosphatidylinositol 4-monophosphate; 1-hexadecanyl-lysophosphatidylinositol 4-monophosphate ⁇ palmityl>; 1-9Z-hexadecenyl-lysophosphatidylinositol 4-monophosphate ⁇ palmitreniinyl>; 1-heptadecanyl-lysophosphatidylinositol 4-monophosphate; 1-octadecanyl-lysophosphatidylinositol 4-monophosphate ⁇ stearyl>; 1-9Z-octadecenyl-lysophosphat
  • acyl (group) is used in the ordinary sense in the art and refers to a group formed by removing a hydroxyl group from an organic acid (carboxylic acid; fatty acid).
  • formyl group HCO—, acetyl group CH 3 CO—, malonyl group —COCH 2 CO—, benzoyl group C 6 H 5 CO—, cinnamoyl group C 6 H 5 CH ⁇ CHCO—, etc. are included, and ketone derivatives And so on.
  • fatty acid groups contained in phosphatidylinositol phosphates and lysophosphatidylinositol phosphates are also referred to as fatty acid groups because they form fatty acids in a preferred embodiment.
  • a fatty acid can be represented by its carbon number and the number of double bonds, for example, arachidonic acid can be represented as (20: 4).
  • arachidonic acid can be represented as (20: 4).
  • fatty acids and acyl groups based thereon are generally used, but the present invention is not limited thereto, and it is understood that fatty acids having any chain length and any double bond can be used.
  • the number of carbon atoms one or more, typically 1 to 30, usually 4 to 30 may be mentioned, 1, 2, 3, 4, 5, 6, 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23 24, 25, 26, 27, 28, 29, 30 and the like, but is not limited thereto.
  • the number of double bonds may be any number that can be allowed according to the number of carbon atoms, such as 0, 1, 2, 3, 4, 5, 6, 7, and the like.
  • the position of the double bond is typically the ⁇ -3 system, ⁇ -6 system, ⁇ -9 system, etc.
  • the ⁇ -5 system, the ⁇ -7 system, etc. have been confirmed. Any available one can be used.
  • the fatty acid may contain a triple bond, and the number thereof is 0, 1, 2, 3, 4, 5, 6, 7, etc. Numbers can be employed.
  • chromatography is used in the ordinary sense used in the art, and when the medium passes (permeates) uniformly through a column or capillary passage of a substance, one or more in the medium. Means a process in which the components of are selectively delayed. The delay is caused by the distribution of the mixture components between one or more stationary phases and this medium as the bulk medium (ie, mobile phase) moves relative to the stationary phase (s).
  • chromatography include liquid chromatography (LC) and gas chromatography (GC). In one embodiment, chromatography can be used to identify the binding position of a phosphate group.
  • gas chromatography or “GC” is used in the ordinary sense used in the art, and when a gas uniformly passes (permeates) through a column or capillary passage of a substance, Means a process in which one or more components are selectively delayed. The delay is caused by the distribution of the mixture components between one or more stationary phases and this gas as the bulk gas (ie, mobile phase) moves relative to the stationary phase (s).
  • liquid chromatography or “LC” is used in the ordinary sense used in the art, and when a fluid passes uniformly (permeates) through a column or capillary passage of matter, Means a process in which one or more components are selectively delayed. The delay is caused by the distribution of the mixture components between one or more stationary phases and this fluid as the bulk fluid (ie, mobile phase) moves relative to the stationary phase (s).
  • liquid chromatography include reverse phase liquid chromatography (RPLC), ion chromatography (IC), high performance liquid chromatography (HPLC), and turbulent liquid chromatography (TFLC) (high turbulent liquid chromatography). (Also referred to as (HTLC) or high-throughput liquid chromatography).
  • HPLC high performance liquid chromatography
  • HPLC also referred to as “high pressure liquid chromatography”
  • HPLC high performance liquid chromatography
  • Separation modes of the filler include normal phase, reverse phase, partition, chiral, ion exchange, molecular sieve, affinity column, etc., and any column chromatography that separates hydrophilic groups and hydrophobic groups can be used. Of these, chiral, reverse phase, normal phase, and ion exchange columns are preferred. In one preferred embodiment, it has been shown in the present invention that when a chiral column is used, all the binding positions of phosphate groups can be identified without using another column.
  • Examples of chiral columns include polysaccharide derivative chiral columns, protein-bonded chiral columns, chemically bonded optically active crown ether chiral columns, crown ether chiral columns, zwitterionic molecular column columns, anion exchange chiral columns, ligand exchange chiral columns, poly Examples thereof include, but are not limited to, methacrylate-type chiral columns and polysaccharide derivative chiral columns (for example, those sold by Daicel Corporation (Osaka, Japan)).
  • the sample or sample components may be applied to the LC column at the inlet, eluted with the solvent or solvent mixture, and discharged at the outlet.
  • Various solvent modes can be selected to elute the analyte (s) of interest.
  • liquid chromatography may be performed using a gradient mode, an isocratic mode, or a polymorphic (ie, mixed) mode.
  • the separation of materials is affected by variables such as the choice of eluent (also referred to as “mobile phase”), elution mode, gradient conditions, temperature, and the like.
  • IMS ion mobility separation
  • a bulkier compound when a gaseous ion passes through an IMS cell filled with N 2 gas, a bulkier compound has more frequent collisions with N 2 molecules and lower mobility.
  • the compounds can be separated by the difference in the transit time (Drift Time) caused thereby.
  • MS mass spectrometry
  • mass spectrometry or “MS” is used in the ordinary sense used in the field, and refers to an analytical method for identifying a compound by its mass, and is a particle such as an atom, molecule, or cluster. Is a technique for separating and detecting ions according to the mass-to-charge ratio by making them into gaseous ions by some method (ionization), moving them in a vacuum and using electromagnetic force, etc., or by a time-of-flight difference. MS refers to a method of filtering, detecting, and measuring ions based on this mass-to-charge ratio, or “m / z”.
  • MS techniques generally include (1) ionizing a compound to form a charged compound: and (2) detecting the molecular weight of the charged compound and calculating a mass to charge ratio.
  • the compound can be ionized and detected by appropriate means.
  • a “mass spectrometer” generally includes an ionizer, a mass analyzer, and an ion detector.
  • the molecule or molecules of interest are ionized and the ions are then introduced into a mass spectrometer where the ions are subject to mass (“m”) and charge (for combination of magnetic and electric fields).
  • m mass
  • charge for combination of magnetic and electric fields
  • z charge
  • Examples of the mass spectrometer include a magnetic field type, a quadrupole type, and a time-of-flight type, and it is preferable to use a quadrupole type that has good quantitativeness, a wide dynamic range, and good linearity.
  • one of the ion species purified by the selected ion monitoring or the first mass analysis unit that selectively detects only the target ions is selected as the precursor ion, and the second ion is detected.
  • selective reaction monitoring (SRM) that detects product ions generated by cleavage of the precursor ions in the mass spectrometer.
  • SRM selective reaction monitoring
  • the term “resolution” or “resolution (FWHM)” is the width of the mass peak at 50% of the maximum height. Refers to the observed mass to charge ratio divided by (full width half maximum, “FWHM”). Also in the present invention, it is preferable to use an analyzer having a high resolution. The higher the resolution, the better the qualitative and quantitative properties.
  • valves and connector piping allows two or more chromatographic columns to be passed as needed so that material passes from one to the next without the need for any manual steps. It may be connected.
  • the selection of valves and piping is controlled by a computer preprogrammed to perform the necessary steps.
  • the chromatography system is also connected to a detection system, such as an MS system, in such an online manner.
  • a detection system such as an MS system
  • C-MS chromatography-mass spectrometry
  • chromatography-mass spectrometry means an analysis method performed using an apparatus combining a chromatograph (eg, gas chromatograph, liquid chromatograph, etc.) and a mass spectrometer. To do.
  • a chromatograph eg, gas chromatograph, liquid chromatograph, etc.
  • mass spectrometer e.g, liquid chromatograph, etc.
  • tandem mass spectrometry C-MS / MS
  • ionization in C-MS for example, atmospheric pressure chemical ionization, ESI, atmospheric pressure photoionization, or the like can be used.
  • liquid column chromatography-mass spectrometry means an analysis method performed using an apparatus in which a liquid chromatograph and a mass spectrometer are combined.
  • tandem mass spectrometry LC-MS / MS
  • mass spectrometry in which a plurality of mass spectrometry units are combined can also be used.
  • ionization in LC-MS for example, atmospheric pressure chemical ionization, ESI, atmospheric pressure photoionization, or the like can be used.
  • IMS-MS ion mobility separation-mass spectrometry
  • IMS-MS means an analysis method performed using an apparatus combining ion mobility separation (IMS) and a mass spectrometer.
  • IMS-MS / MS tandem mass spectrometry
  • IMS-MS ion mobility separation-mass spectrometry
  • MS-MS tandem mass spectrometry
  • IMS-MS enables separation of interfering components having the same m / z, which is impossible with a mass spectrometer alone, and analysis of the three-dimensional structure of ions, and can provide more specific and detailed information to scientists.
  • measurement is used in the normal sense used in the field, and refers to determining how much a certain object is measured.
  • detection is used in the usual meaning used in the field, refers to finding out a substance, component, etc.
  • identity refers to an existing object related to itself. This refers to the act of finding the attribution of the classification from the classification of the substance, and when used in the chemical field, it refers to determining the identity of the target substance as a chemical substance (for example, determining the chemical structure), “Quantitative” refers to determining the amount of a target substance.
  • the term “run” is used in the ordinary meaning used in the art, and the sample is loaded in a separation means such as mass spectrometry, chromatography, and ion mobility separation to separate components in the sample. , Refers to a series of steps until washing as necessary. Usually, different samples are measured in different runs to avoid confusion. When there are multiple measurement objects, it is advantageous to be able to measure all measurement objects in the same run, but the run is considered in consideration of adaptability to the measurement system for each measurement object in the sample, dynamic range, and separation degree. You may divide into several.
  • the “amount” of an analyte in a body fluid sample generally refers to an absolute value that reflects the mass of the analyte that can be detected in the volume of the sample. However, an amount also contemplates a relative amount compared to another analyte amount. For example, the amount of analyte in the sample may be an amount greater than the control or normal level of analyte normally present in the sample.
  • alkylamine refers to an alkyl group to which an amine (NH 2 —) group is bonded. Typical examples include, but are not limited to, methylamine, ethylamine, propanamine and the like. Also referred to as aliphatic amine or aminoalkane.
  • anion exchange resin is used in the ordinary sense in the art, and typically has a property in which a basic group is bonded to the surface of the resin and binds to an anion. Can be concentrated.
  • acidic phospholipid refers to a phospholipid in which the negative charge of the phosphate group is not canceled by a base, and includes phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol ( PI).
  • PA phosphatidic acid
  • PS phosphatidylserine
  • PI phosphatidylinositol
  • protecting group for the phosphate group, any substance used in the art can be used.
  • Protecting groups are exemplified in, for example, Clark J et al, Nat Methods. 2011 March; 8 (3): 267-272.doi: 10.1038 / nmeth.1564.
  • Benzyl group, p-methoxybenzyl group, Examples thereof include a tert-butyl group.
  • the conditions under which phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate are not decomposed means a condition in which components such as acyl groups are not decomposed and eliminated from phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate.
  • “Conditions under which phosphatidylinositol phosphate is not decomposed” refers to conditions in which components such as acyl groups are decomposed from phosphatidylinositol phosphate and do not leave, and “Conditions under which lysophosphatidylinositol phosphate is not decomposed” refers to lysophosphatidyl.
  • the conditions under which components such as acyl groups are not decomposed and eliminated from inositol phosphate, and the conditions under which phosphatidylinositol phosphate and lysophosphatidylinositol phosphate are not decomposed are the conditions under which phosphatidylinositol phosphate and lysophosphatidylinositol phosphate This is a condition in which components such as are decomposed and do not desorb.
  • Such conditions can be applied to chromatography, ion mobility separation, or MS by applying other treatments under conditions that do not include deacylation, in addition to applying the sample directly to chromatography, ion mobility separation, or MS.
  • the treatment of denaturing the sample is not performed, and the treatment of introducing a protecting group into the phosphate group is included.
  • no deacylation treatment means deacylation, for example, treatment with phospholipases A (phospholipase A 1 , A 2 etc.), treatment in the presence of alkylamine such as methylamine ( Serunian, et al., Methods in Enzymol. Vol., 198, 1991, pp. 78-87, especially pp. 82-83, and Fujii et al. Folia Pharmacol Jpn. 2013, pp. 236-240, especially pp. 238-239).
  • alkylamine such as methylamine
  • sample means that when a sample to be analyzed is assumed, the sample is not denatured.
  • sample denaturation include denaturation by heating, oxidation in the air, moisture, heat, light, metal ions, microorganisms or enzymes, and hydrolysis in an aqueous solvent. In order to be in an intact state, for example, the sample is frozen immediately after preparation and stored at ⁇ 80 ° C. until the subsequent operation is performed.
  • label refers to a presence (for example, a substance, energy, electromagnetic wave, etc.) for distinguishing a target molecule or substance from others.
  • a labeling method include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method and the like.
  • the labeling is performed with fluorescent substances having different fluorescence emission maximum wavelengths.
  • the target object can be modified so that it can be detected by the detection means used. Such modifications are known in the art, and those skilled in the art can appropriately carry out such methods depending on the label and the target object.
  • diagnosis identifies various parameters related to a disease, disorder, condition (eg, a disease, disorder, condition, etc. caused by a lipid mediator) in a subject, and such a disease, disorder , Refers to determining the current state or future of the state.
  • conditions within the body can be examined, and such information can be used to formulate a disease, disorder, condition, treatment to be administered or prevention in a subject.
  • various parameters such as methods can be selected.
  • diagnosis in a narrow sense means diagnosis of the current state, but in a broad sense includes “early diagnosis”, “predictive diagnosis”, “preliminary diagnosis”, and the like.
  • the diagnostic method of the present invention is industrially useful because, in principle, the diagnostic method of the present invention can be used from the body and can be performed away from the hands of medical personnel such as doctors.
  • “predictive diagnosis, prior diagnosis or diagnosis” may be referred to as “support”.
  • the technique of the present invention can be applied to such a diagnostic technique.
  • the presence of phosphoinositide and / or lysophosphoinositide or the position of the phosphate group thereof can be specified and applied to such various diagnoses.
  • treatment refers to a certain disease or disorder (for example, a disorder such as cancer, a disease caused by a lipid mediator, a disorder, etc.), when such a condition or Prevents the deterioration of the disorder, preferably maintains the current state, more preferably reduces, and even more preferably eliminates the disorder, or exhibits the symptom improving effect or preventing effect of one or more symptoms associated with the disease. It includes what can be done. Diagnosing in advance and performing appropriate treatment is referred to as “companion treatment”, and the diagnostic agent therefor is sometimes referred to as “companion diagnostic agent”.
  • the ability to identify the presence of phosphoinositide and / or lysophosphoinositide or the binding position of its phosphate group using the techniques of the present invention can be associated with a particular disease state and thus such companion therapy or companion diagnosis May be useful.
  • prevention refers to preventing a certain disease or disorder (for example, cancer, a disease or disorder related to lipid mediators, etc.) from entering such a state before such a state occurs. That means. Possibility that diagnosis can be performed using the PIPs and / or LPIPs of the present invention, and if necessary, for example, prevention of diseases or the like can be taken using the drug of the present invention. There is.
  • prolactic agent refers to any agent that can prevent a target condition (for example, cancer, diseases and disorders related to lipid mediators, etc.).
  • prognosis means predicting the possibility of death or progression due to a disorder such as cancer, a disease caused by a lipid mediator, or a disorder.
  • Prognostic factors are variables related to the natural course of the disease, and these affect the recurrence rate of patients who have once developed the disease.
  • Clinical indicators associated with worse prognosis include, for example, any cellular indicator used in the present invention.
  • Prognostic factors are often used to classify patients into subgroups with different pathologies.
  • phosphatidylinositol and / or lysophosphatidylinositol or the binding position of its phosphate group can be identified using the technology of the present invention, it is useful as a technology for providing a prognostic factor because it can be associated with a specific disease state. obtain.
  • detection device means, in a broad sense, any device that can detect or inspect a target object.
  • a chromatography, an ion mobility separation device, a mass analyzer, and the like are also included in the detection device.
  • diagnosis device means, in a broad sense, any drug capable of diagnosing a target condition (for example, cancer, diseases and disorders related to lipid mediators, etc.).
  • drug drug
  • drug may also be a substance or other element (eg energy such as light, radioactivity, heat, electricity).
  • Such substances include proteins (including antibodies, etc.), polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (for example, DNA such as cDNA and genomic DNA, mRNA and the like) RNA), polysaccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals, etc.)
  • proteins including antibodies, etc.
  • polypeptides for example, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (for example, DNA such as cDNA and genomic DNA, mRNA and the like) RNA), polysaccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, lig
  • detection agent or “test agent (agent)” refers to any agent that can detect or inspect a target object in a broad sense.
  • diagnostic agent broadly refers to any agent capable of diagnosing a target condition (for example, cancer, diseases and disorders related to lipid mediators, etc.).
  • therapeutic agent refers to any agent that can treat a target condition (for example, cancer, diseases or disorders related to lipid mediators, etc.).
  • cancer refers to any cancer detectable with the marker of the present invention, such as hepatocellular carcinoma, squamous cell carcinoma of the esophagus, breast cancer, pancreatic cancer, and squamous cells of the head and neck.
  • inflammation refers to any inflammation detectable with the marker of the present invention, and activation of blood cells related to inflammation such as macrophages (inflammatory cell chemotaxis, active oxygen production, phagocytosis, enzyme A secretory reaction, etc.).
  • macrophages inflammatory cell chemotaxis, active oxygen production, phagocytosis, enzyme A secretory reaction, etc.
  • Exemplary inflammations include, for example, arthritis, tendinitis, bursitis, psoriasis, cystic fibrosis, Sjogren's syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), spondylitis, multiple Dermatomyositis, dermatomyositis, pemphigus, pemphigoid, Hashimoto's thyroiditis, cholangitis, inflammatory bowel disease (IBD, eg Crohn's disease, ulcerative colitis), colitis, inflammatory skin disease, pneumonia, asbestosis, Silicosis, bronchiectasis, talc lung, pneumoconiosis, sarcoidosis, delayed hypersensitivity reaction (eg poison ivy dermatitis), respiratory tract inflammation, adult respiratory distress syndrome (ARDS), encephalitis, immediate hypersensitivity reaction, asthma, hay fever, allergy , Acute anaphylaxis, reperfusion injury, rheumato
  • the “cancer marker” is also referred to as a tumor marker, and refers to a biological substance that is effective for cancer diagnosis, follow-up after treatment, discovery of recurrence or metastasis, or a substance found in the living body.
  • cancer diagnosis and the like can be performed by measuring substances in the blood.
  • the substance in the blood corresponds to the cancer marker.
  • the term “marker” is in a certain state (for example, functionality, transformation state, disease state, disorder state, proliferative ability, level of differentiation state, presence / absence, etc.) or is there a risk thereof?
  • “disease marker” refers to a substance that serves as an indicator for tracking whether a disease state is present or at risk.
  • detection, diagnosis, preliminary detection, prediction, or prior diagnosis for a certain state for example, a disease state, a health state, a disease such as a cell or tissue differentiation disorder
  • it can be realized by using a marker-specific drug, agent, factor or means, or a composition, kit or system containing the same.
  • a “purified” substance or biological agent refers to an agent that naturally accompanies the substance or biological agent. This means that at least a part has been removed. Thus, typically, the purity of a biological agent in a purified biological agent is higher (ie, enriched) than the state in which the biological agent is normally present.
  • the term “purified” as used herein is preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight, Means the presence of the same type of biological agent.
  • the substance or biological agent used in the present invention is preferably a “purified” substance.
  • an “isolated” substance or biological agent such as a nucleic acid or protein
  • the term “isolated” as used herein does not necessarily have to be expressed in purity, as it will vary depending on its purpose, but is preferably at least 75% by weight, more preferably if necessary. Means that there is at least 85%, more preferably at least 95%, and most preferably at least 98% by weight of the same type of biological agent.
  • the materials used in the present invention are preferably “isolated” materials or biological agents.
  • compositions, medicaments, agents (therapeutic agents, prophylactic agents, etc.) of the present invention comprise a therapeutically effective amount of a medicament or active ingredient, and a pharmaceutically acceptable carrier or excipient.
  • pharmaceutically acceptable refers to a licensed or otherwise recognized pharmacopoeia of a government for use in animals, and more particularly in humans, by a government supervisory authority. It means that it is enumerated.
  • a “patient” is mainly assumed to be a human, but may be a mammal other than a human as long as it is applicable.
  • subject refers to a subject to be used for the prevention or treatment of the present invention, and is a human or a mammal other than a human (eg, mouse, guinea pig, hamster, rat, rat, Including one or more of a rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset, monkey, or chimpanzee).
  • the “kit” is a unit provided with a portion to be provided (eg, a test agent, a diagnostic agent, a therapeutic agent, a reagent, a label, an instruction, etc.) usually divided into two or more compartments.
  • a portion to be provided eg, a test agent, a diagnostic agent, a therapeutic agent, a reagent, a label, an instruction, etc.
  • This kit form is preferred when it is intended to provide a composition that should not be provided in admixture for stability or the like, but preferably used in admixture immediately before use.
  • Such kits preferably include instructions describing how to use or how to handle the provided moiety (eg, test, diagnostic, therapeutic, reagent, label, etc.).
  • kit when used as a reagent kit, the kit describes how to use a test agent, a diagnostic agent, a therapeutic agent, a reagent, a label, and the like. This includes instructions, etc.
  • a “kit” can be provided as a “system”.
  • the “instruction sheet” describes the method for using the present invention for a doctor or other user.
  • This instruction manual includes a word indicating that the detection method of the present invention, how to use a diagnostic agent, or administration of a medicine or the like is given.
  • the instruction sheet may include a word indicating a method for detecting and determining the marker.
  • This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc. in the United States) where the present invention is implemented, and is approved by the supervisory authority. It is clearly stated that it has been received.
  • the instruction sheet is a so-called package insert and is usually provided in a paper medium, but is not limited thereto, and is in a form such as an electronic medium (for example, a homepage or an e-mail provided on the Internet). But it can be provided.
  • LPIPs ⁇ Lysophosphatidylinositol phosphates (LPIPs)>
  • the present invention relates to lyso-phosphatidylinositol monophosphate (LPIP1), lysophosphatidylinositol diphosphate (LPIP2) or lysophosphatidylinositol triphosphate (lyso-phosphatidylinositol monophosphate (LPIP2)).
  • LPIP3 phosphatidylinositol trisphosphate
  • Phosphoinositide / inosyl phospholipids are said to constitute plasma membranes and organelle membranes, and their head group is significantly larger than other glycerophospholipids due to the six-membered carbon ring and polyvalent phosphate groups.
  • the PIPs metabolic system rich in electric charge is considered to be involved in a wide range of life phenomena such as proliferation, differentiation, movement, aging, and death.
  • the lipids (LPIPs) of the present invention identified for the first time in the present invention can be useful as targets for various therapeutic drugs and as disease markers. Since lipid metabolism is one of the important research subjects, these lipids are used as experimental reagents as reagents.
  • LPIPs lyso form of PIPs
  • an anion exchange resin such as DEAE cellulose
  • TMS diazomethane protected phosphate groups with TMS diazomethane
  • SRM method with a triple quadrupole mass spectrometer.
  • Analytical method was developed. By using this method, the inventors succeeded in finding LPIP1, LPIP2, and LPIP3 of the present invention, which are structures different from known inositol phospholipids. Also, LPIPs could not be produced because the synthesis method was not known.
  • LPIPs have not been confirmed as substances and can be said to have not been known.
  • synthetic methods can be developed, and in addition to providing LPIPs existing in vivo, LPIPs including “non-natural type” LPIPs can be provided by synthesis.
  • LPIPs are also shown in the present invention to be present in vivo.
  • it is found in various mammalian cell lines and mouse prostate cancer tissues, and seems to be preferentially expressed in cancer cells in specific molecular species such as LPIP3. It became clear. It was also found that some of the found LPIPs are specifically or preferentially expressed in cancer.
  • PIPs having a structure similar to LPIPs have been analyzed in the past. For example, it is known that mutations and decreased expression of the tumor suppressor gene PTEN are abnormalities that are frequently observed in about half of human cancers. ing.
  • LPIPs of the present invention are The possibility of working as a lipid mediator can also be considered. Moreover, it was found by high sensitivity measurement that LPIPs exist not only in cells but also outside cells. Measuring these lipids with a sample that is relatively easy to collect, such as blood and urine, is also useful in laboratory medicine.
  • LPIPs have been found as novel substances in the present invention, and it has been found that some LPIPs are preferentially expressed in cancer, blood, urine in diseases associated with cancer or other lipid mediators It can be applied to medical examinations and diagnosis using specimens such as.
  • phospholipases A phospholipases A 1 , A 2, etc.
  • phospholipases A 1 and A 2 have extremely high substrate specificity, It does not respond to different types of phospholipids.
  • a subtype having phosphatidylcholine as a substrate does not react with phosphatidylserine.
  • PIPs as a substrate is not known.
  • lysophosphatidylinositol monophosphate lysophosphatidylinositol diphosphate
  • lysophosphatidylinositol triphosphate have been found.
  • lysophosphatidylinositol monophosphate found in the living body, a phosphate group is located at the 4-position, and a species in which the following fatty acids are bonded to the sn-1 position or the sn-2 position was found.
  • fatty acid molecular species are similar to the fatty acid species found in PIPs, suggesting the presence of a previously unknown phospholipase A 1 or phospholipase A 2 that has specificity for phosphatidylinositol phosphates.
  • the fatty acid molecular species are likely to be found in both sn-1 and sn-2 in vivo, although there are many abundances.
  • the invention provides 1-acyl-lysophosphatidylinositol monophosphate, 2-acyl-lysophosphatidylinositol monophosphate, 1-acyl-lysophosphatidylinositol diphosphate, 2-acyl-lysophosphatidylinositol.
  • Compounds are provided that are diphosphate, 1-acyl-lysophosphatidylinositol triphosphate, or 2-acyl-lysophosphatidylinositol triphosphate.
  • the acyl group may be any kind of acyl group as long as the corresponding fatty acid is available.
  • the lysophosphatidylinositol phosphates of the present invention can be produced starting from phosphatidylinositol phosphates. Therefore, by introducing the obtained fatty acid into phosphatidylinositol phosphates based on the Conway method cited in this specification, it can be used as a starting material for the production method of lysophosphatidylinositol phosphates of the present invention.
  • the present invention provides 2-acyl-lysophosphatidylinositol monophosphate, 2-acyl-lysophosphatidylinositol diphosphate, 1-acyl-lysophosphatidylinositol triphosphate or 2-acyl-lysophosphatidyl.
  • a compound that is inositol triphosphate is provided.
  • lysophosphatidylinositol phosphates can be used in which the phosphate group is bonded to any position in inositol, and preferably one of 2, 3, and 5 positions. Or what was couple
  • the present invention provides 1-acyl-lysophosphatidylinositol-3-monophosphate, 1-acyl-lysophosphatidylinositol-4-monophosphate, 1-acyl-lysophosphatidylinositol-5-monophosphate Acid, 2-acyl-lysophosphatidylinositol-3-monophosphate, 2-acyl-lysophosphatidylinositol-4-monophosphate, 2-acyl-lysophosphatidylinositol-5-monophosphate, 1-acyl-lysophosphatidyl Inositol-3.4-diphosphate, 1-acyl-lysophosphatidylinositol-3.5-diphosphate, 1-acyl-lysophosphatidylinositol-4.5-diphosphate, 2-acyl-lysophosphatidylinositol- 3,4-diphosphate, 2-
  • the present invention illustratively provides the following compound, but the specific compound of the present invention is not limited to the following.
  • the present invention provides a composition for use as a disease marker comprising a compound of the present invention (such as lysophosphatidylinositol phosphates).
  • a compound of the present invention such as lysophosphatidylinositol phosphates. It is understood that as such a compound, any embodiment described in ⁇ lysophosphatidylinositol phosphates (LPIPs)> in this specification may be employed in combination of one or more.
  • LPIP1 lysophosphatidylinositol monophosphate
  • LPIP2 lysophosphatidylinositol diphosphate
  • LPIP3 lysophosphatidylinositol triphosphate
  • LPIP3 has been shown to have a significantly increased expression level in cancer tissues, and the compound of the present invention should be used as an inflammation marker or a cancer marker, particularly in the case of substances present in vivo. It is understood that a synthetic product is also useful as a control or standard for detection of an inflammatory marker or a cancer marker.
  • the target cancer is not limited.
  • the target inflammation is not limited, for example, arthritis, tendinitis, bursitis, psoriasis, cystic fibrosis, Sjogren's syndrome, giant cell arteritis , Progressive systemic sclerosis (scleroderma), spondylitis, polymyositis, dermatomyositis, pemphigus, pemphigoid, Hashimoto's thyroiditis, cholangitis, inflammatory bowel disease (IBD such as Crohn's disease, ulcerative) Colitis), colitis, inflammatory skin disease, pneumonia, asbestosis, silicosis, bronchiectasis, talc lung, pneumoconiosis, sarcoidosis, delayed type hypersensitivity reaction (eg poison ivy dermatitis), airway inflammation, adult respiratory distress syndrome (ARDS), encephalitis, immediate hypersensitivity reaction, asthma, hay fever, allergy, acute anaphylaxis
  • IBD inflammatory bowel disease
  • colitis
  • the LPIPs of the present invention can be used for the detection method and other methods disclosed in the present specification (for example, immunological techniques, cytological techniques using radioactive substances, etc.) ) Can be used.
  • radioactive materials intracellular LPIPs labeled with radioactive isotopes (RI) following the PIPs method (see Sasaki et al., Ayumi Sci. Vol. 248, No. 13, (2014) pp. 1039-1049) Are separated by high performance liquid chromatography (HPLC) to obtain radioactivity.
  • the detection, identification, quality control, and the like of the LPIPs of the present invention can be realized by using a substance or an interacting molecule that binds to LPIPs serving as a marker, in addition to using the detection method described in the present invention.
  • a “substance that binds” or “interacting molecule” to a marker substance binds and preferably binds to a molecule, such as a substance that is at least temporarily marker (eg, LPIPs).
  • a molecule or substance that can indicate eg, is labeled or is ready to be labeled).
  • Substances that bind to molecules such as LPIPs may be receptors for molecules such as LPIPs or transferases (if known), and other examples include antibodies, antisense oligonucleotides, siRNAs, low molecular weight molecules ( LMW), binding peptides, aptamers, ribozymes, peptidomimetics, and the like, including, for example, binding proteins specific for molecules such as LPIPs.
  • binding protein for a molecule such as LPIPs refers to a type of protein that binds (preferably specifically) to a molecule such as LPIPs, and is induced to a molecule such as LPIPs. Including but not limited to antibodies such as polyclonal or monoclonal antibodies, antibody fragments and protein backbones. A typical example of such a protein is an antibody, and such an antibody can be carried out using a technique known in the art.
  • the present invention relates to a compound of the present invention (lysophosphatidylinositol) comprising the step of deacylating phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate by contacting with alkylamine. (Phosphate) production method.
  • the alkylamine is methylamine
  • the deacylation is carried out at a temperature (eg, room temperature to about 80 ° C.) at which the deacylation promotes in the presence of an appropriate concentration (eg, 10.7% or less) of an alkylamine such as methylamine. Incubate for an appropriate period of time (typically, about 53 ° C. can be used.)
  • a temperature eg, room temperature to about 80 ° C.
  • an appropriate concentration eg, 10.7% or less
  • an alkylamine such as methylamine.
  • Incubate for an appropriate period of time typically, about 53 ° C. can be used.
  • Serunian, et al. Methods in Enzymol disclosing the dediacylation conditions Vol., 198, 1991, pp. 78-87, especially pp. 82-83, Fujii et al. Folia Pharmacol Jpn. Vol. 142, 2013, pp. 236-240, especially pp. 238-239.
  • the monoacyl is unexpectedly cleaved by using it for a short period of time such as about 30 minutes or less, and sn-1
  • LPIPs or sn-2 LPIPs are generated.
  • the concentration of methylamine was 2.13%, only monoacyl was cleaved unexpectedly under the reaction conditions of 120 minutes to produce LPIP.
  • deacylation means that the fatty acid bond at the 1-position and / or 2-position in phosphatidylinositol phosphate (which may be monophosphate, diphosphate, or triphosphate, also referred to as PIPs). It means that the acyl group is cleaved and cleaved (eliminated). Unless otherwise specified herein, “deacylation” includes “demonoacylation” in which one acyl group is cleaved (eliminated) and “dediacylation” in which two acyl groups are cleaved (eliminated). Both reactions can be included. As used herein, it includes “demonoacylation”. “Demonoacylation” can be either of the sn-1 position or of the sn-2.
  • methylamine / H 2 O / methanol / n-butanol 1.92 / 38.08 / 40/10 (deacylation reaction solution) was added to the dried PIPs, and 53 Incubate at 5 ° C for 5 minutes. An ice-cooled deacylation reaction solution is added to stop the reaction (an exemplary result obtained by allowing to stand on ice for 1 minute and then drying to measure the product is shown in FIG. 1).
  • One or more (or all) of various conditions such as solvent composition, reaction temperature, reaction time, catalyst (typically methylamine) concentration, etc. may be appropriately changed as long as demonoacylation occurs. can do.
  • the object of the present invention can be achieved if the amount of methylamine is 1-3%.
  • phosphatidylinositol monophosphate phosphatidylinositol diphosphate or phosphatidylinositol triphosphate
  • phosphatidylinositol monophosphate phosphatidylinositol diphosphate or phosphatidylinositol triphosphate
  • any kind of PIP can be manufactured by Stuart J. Conway et al., Org. Biomol. Chem., 2010, 8, 66-76.
  • the present invention provides (A) providing phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate having different mass acyl groups at the 1-position and 2-position, An acyl group having a desired mass is present at a desired position (position 1 or position 2); (B) the phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate with an alkylamine Deacylation by contact with (usually performed under the demonoacylation conditions described herein), and (C) lysophosphatidylinositol monophosphate having a desired mass of acyl groups, lyso Phosphatidylinosito
  • phosphatidylinositol monophosphate, phosphatidylinositol diphosphate or phosphatidylinositol triphosphate having different masses of acyl groups at the 1-position and the 2-position are obtained from Stuart J. Conway et al., Org. Biomol. Chem., 2010, 8, 66-76 can be used as a reference and will be described in detail below.
  • deacylation the above-mentioned conditions can be appropriately applied.
  • the desired lysophosphatidylinositol monophosphate, lysophosphatidylinositol diphosphate or lysophosphatidylinositol triphosphate can be removed by any means that can be separated by differences in mass, hydrophobicity, and the like. For example, chromatography can be used. Of course, the purification step is not essential if it can be used without purification.
  • R 1 and R 2 are each independently any fatty acid residue, and R B and R C are each independently hydrogen or an alkyl group (for example, 1 to 12 carbon atoms such as methyl, ethyl, etc. an alkyl group) and the like
  • PG a, PG P1 and PG P2 as are each independently a protecting group. protecting groups such as benzyl group, p- methoxybenzyl group, be mentioned tert- butyl group (However, it is not limited to these.
  • compound P-2 By subjecting compound P-2 to appropriate conditions (for example, by using trifluoroacetic acid), the protecting group PG P1 (for example, methoxymethyl group) of the hydroxy group is deprotected to obtain compound P-3.
  • compound P-3 and a carboxylic acid halide are subjected to appropriate conditions (for example, dimethylaminopyridine (DMAP) and octadecanoyl chloride are added to a dichloromethane solution of compound P-3 at 0 ° C. and stirred at room temperature.
  • DMAP dimethylaminopyridine
  • octadecanoyl chloride are added to a dichloromethane solution of compound P-3 at 0 ° C. and stirred at room temperature.
  • Compound P-4 is obtained.
  • compound P-4 By subjecting compound P-4 to appropriate conditions (for example, by using 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) or ammonium hexanitrerium (IV) acid (CAN))
  • the protecting group PG P2 for example, 4-methoxybenzyl group
  • compound P-5 Compound 1-5 and aminophosphine (eg (4-methoxybenzyloxy) bis (N, N-diisopropylamino) phosphine) are subjected to appropriate conditions (eg 1H-tetrazole is added at room temperature in dichloromethane solution).
  • aminophosphine eg (4-methoxybenzyloxy) bis (N, N-diisopropylamino) phosphine
  • PG 1 , PG A , and PG 2 are each independently a protecting group.
  • the protecting group include, but are not limited to, methoxybenzyl and the like.
  • Compound I-1 with subjecting to suitable conditions e.g. the compound I-1 of DMF was added 4-methoxybenzyl chloride and sodium hydride was added at 0 ° C., by stirring at room temperature
  • protecting group PG A Compound I-2 into which (for example, 4-methoxybenzyl group) is introduced is obtained.
  • compound I-2 By subjecting compound I-2 to appropriate conditions (for example, by adding chloromethyl methyl ether and sodium hydride to a DMF solution of compound I-2 at 0 ° C.
  • protecting group PG 1 For example, compound I-3 into which a methoxymethyl group is introduced is obtained.
  • compound I-3 By subjecting compound I-3 to appropriate conditions (for example, by adding diisobutylaluminum hydride (DIBAL) to a dichloromethane / hexane solution of compound I-3 at 0 ° C. and stirring at room temperature), compound I-4 obtain.
  • compound I-4 By subjecting compound I-4 to appropriate conditions (for example, by adding 4-methoxybenzyl chloride and sodium hydride to a DMF solution of compound I-4 at 0 ° C. and stirring at room temperature), the protecting group PG 2 Compound (I-5) into which (for example, 4-methoxybenzyl group) is introduced is obtained.
  • compound I-6 By subjecting compound I-5 to appropriate conditions (for example, adding hydrochloric acid to a methanol solution of compound I-5 and refluxing), compound I-6 is obtained. By subjecting compound I-6 to appropriate conditions (for example, a solution of compound I-6 in dichloromethane with toluenesulfonic acid monohydrate)
  • compound I-8 (l, l) having 1 hydroxy group, m hydroxy groups protected with protecting group PG 1 and n hydroxy groups protected with PG 2 is obtained.
  • m and n are integers of 0 or more and 6 or less, and the sum of l, m, and n is 6.
  • Suitable for the hydroxy group of inositol precursor PI-1 eg 1D-2,3,4,5,6-penta-O- (4′-methoxybenzyl) -1-O- (methoxymethyl) -myo-inositol
  • a suitable phosphorus reagent eg bis (4-methoxybenzyloxy) (N, N-diisopropylamino) phosphine
  • PI-2 modified with a phosphate group (for example, 1D-2,3,4,5,6-penta-O- (4′-methoxybenzyl) -1-) by stirring at room temperature for 10 to 14 hours O- (methoxymethyl) -myo-inositol-4
  • the protective group PG 1 for example, methoxymethyl group
  • PI-3 for example, 1D-2,3,4,5, 6-Penta-O- (4′-methoxybenzyl) -myo-inositol-4- (bis (4-methoxybenzyloxy) phosphate)
  • PI-3 and a phosphoramidite precursor eg (1-acetyloxy-2′-octadecanoyloxypropyl) (4-methoxybenzyl) diisopropyl phosphoramidite
  • the appropriate conditions eg PI— 1-tetrazole and (1-acetyloxy-2-octadecanoyloxypropyl) (4-methoxybenzyl) diisopropyl phosphoramidite
  • metachloroperbenzoic acid PI-4 is obtained by adding acid (mCPBA) at ⁇ 78 ° C. and stirring for 20 minutes at room temperature.
  • the protective group PG 2 (eg 4-benzyl) is added to PI-4 under appropriate conditions (for example, by adding ammonium hexanitracelium (IV) to a solution of PI-4 in acetonitrile and stirring for 45 minutes at room temperature).
  • PI-5 eg, 1D-myo-inositol-1- (1′-O-acetyl-2′-O-octadecanoyl-sn-glycera-3-yl phosphate) is obtained by deprotecting the group.
  • Phosphatidylinositol phosphate produced by the above production method includes, for example, the following formula
  • a solution of phosphatidylinositol 1 (eg 1D-myo-inositol-1- (1′-O-acetyl-2′-O-octadecanoyl-sn-glycera-3-yl phosphate) (eg chloroform / methanol (9 / 1)
  • a solution may be mentioned, but the ratio of the solvent for preparing the solution can be appropriately changed, and another solvent can be used as long as the reaction is promoted.) Is dried with an inert gas such as N 2.
  • a suitable reagent for example, but not limited to, a methylamine solution
  • solvents include methylamine / water / methanol / 1-butanol (4.8 / 35.2 / 40/10).
  • the ratio of the solvent for the preparation and methylamine can be appropriately changed, and another solvent can be used as long as the reaction is accelerated. It can be added at a temperature (eg, 53 ° C.) and exemplified for a suitable time (eg, 5 minutes), but can be stretched as long as it is longer or shorter as long as demonoacylation is achieved. It has been observed that demonoacylation occurs in 30 seconds.) Suitable temperatures (eg, 53 ° C.
  • COR 1 is an acetyl group
  • COR 2 If a octadecanoyl group), 2 and 3 acyl group is a compound desorbed only one, since the molecular weight are different, it is possible to separate and purify each compound.
  • the present invention provides a method for detecting, identifying or quantifying lysophosphatidylinositol phosphate, wherein (A) concentrating a phospholipid by contacting a sample with an anionic resin; B) protecting the phosphate group in the phospholipid with a protecting group; and (C) detecting, identifying or quantifying lysophosphatidylinositol phosphate in the phospholipid by mass spectrometry. Methods for detecting, identifying or quantifying phospholipids are provided.
  • Phosphoinositides including lysophosphatidylinositol phosphate
  • the method of the present invention was able to set several hundred amol to several fmol as the lower limit of quantification, and was able to measure many types of LPIPs that were unknown whether they existed in vivo.
  • the present invention provides a mass spectrometry based method capable of absolute quantification with various biological trace samples.
  • the present invention provides for the first time a technique for identifying and detecting lysophosphatidylinositol phosphates.
  • any protecting group for the phosphate group of the present invention can be used as long as it can be protected, but preferably it does not become an obstacle in mass spectrometry or is desorbed before mass spectrometry.
  • an alkyl group for example, a methyl group
  • the protecting group contains an alkyl group, more preferably a methyl group.
  • any technique can be used for the mass spectrometry used in the present invention, it preferably includes a selective reaction monitoring method (SRM) using a triple quadrupole mass spectrometer.
  • SRM selective reaction monitoring method
  • the SRM used in the present invention further includes a step of detecting, identifying or quantifying the fatty acid side chain and / or phosphate group of the lysophosphatidylinositol phosphate using reverse phase column chromatography.
  • diacylglycerol in the reverse phase column chromatography, can be selected as a product ion (fragment ion) to detect, identify or quantify the fatty acid side chain and / or phosphate group.
  • PIP1 and PIP2 are each quantified as the sum of three types of isomers (structural isomers with different positions of phosphorylated hydroxyl groups). The procedure is outlined below.
  • the total lipid fraction obtained by the high recovery Bligh-Dyer method of phosphoinositide such as lysophosphatidylinositol phosphates in vivo is further fractionated using an anion exchange column (DEAE cellulose column). Gradually separate and elute according to the difference in ion exchange capacity of each phospholipid polar site, and fractions rich in phosphoinositide can be recovered at a high rate, and lysophosphatidylinositol phosphates should be recovered at a high rate as well. Can do.
  • lysophosphatidylinositol phosphates are also the same. It is thought that it has the property of. Therefore, phosphoinositide is rapidly damaged in the analytical vial during the waiting time from collection to measurement, and tailing is seen in the detected elution peak.
  • the present inventors perform a robust analysis by producing a stable derivative that suppresses decomposition / adsorption by methylating a phosphate group after high recovery of phosphoinositide. Such a technique can also be applied to lysophosphatidylinositol phosphates.
  • a selective reaction monitoring method using a triple quadrupole mass spectrometer, which is a highly sensitive and highly quantitative analytical method, is used.
  • SRM selective reaction monitoring method
  • Phosphoinositide is easy to detect as a negative ion due to its structure, but in the analysis method of the present invention, it is easy to detect as a positive ion because the phosphate group is protected such as methylation.
  • Lysophosphatidylinositol phosphates also show similar properties. Each molecular species selects diacylglycerol (DG) as a characteristic fragment ion.
  • DG diacylglycerol
  • Analysis is carried out by reverse phase column (C8) using the difference in hydrophobicity of fatty acid side chains to separate and elute each molecular species.
  • Each phosphoinositide of the same molecular species elutes in the order of PIP3, PIP2, and PIP1 due to the difference in the number of phosphate groups, and therefore, analysis is performed using this difference in elution time.
  • the peak area of the chromatogram of each molecular species that has been smoothed by the Gaussian method is digitized, and this is applied to a calibration curve prepared using various 17: 0/20: 4 synthetic products that are hardly present in the living body. To calculate.
  • This analysis method of the present invention is highly sensitive and excellent in quantification, and can be analyzed with human clinical specimens and animal tissue-derived samples in addition to cultured cells. The same analysis can be performed for lysophosphatidylinositol phosphates. In addition to the differences in molecular weight, LPIP3, LPIP2, and LPIP1 are eluted in this order. The peak area of the chromatogram of each molecular species subjected to smoothing can be digitized, compared with a standard product, and applied to the prepared calibration curve for calculation.
  • the present invention provides a technique for distinguishing and detecting lysophosphatidylinositol phosphates, which can also be disease markers, from other substances, it can also diagnose cancer.
  • LPIPs diacyl-type inositol phospholipids
  • the LPIPs of the present invention which are structurally closely related to diacyl-type inositol phospholipids (PIPs) are also known to be related to various pathological conditions, and have usefulness as therapeutic targets and diagnostic markers. Expected to have. Specifically, the following uses are assumed. Also, Biochemistry Vol. 83, No. 6, pp.
  • lysophosphatidylinositol As described in 525-535, 201, it is known to have various activities including lysophosphatidylinositol as a lipid mediator and a G protein receptor such as its receptor GPR55. Therefore, the compound of the present invention in which lysophosphatidylinositol is phosphorylated in various forms is expected to be directly or indirectly related to various physiological uses in relation to lysophosphatidylinositol and its receptor.
  • the inositol polyphosphate group produced from PIPs by the action of phospholipase C controls intracellular calcium kinetics and the like.
  • the LPIPs of the present invention that are structurally closely related to the inositol polyphosphate group are also expected to have utility as therapeutic targets and diagnostic markers.
  • the present invention provides a method for measuring, detecting or identifying phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate in a sample.
  • the method comprises A) applying the sample to mass spectrometry (MS); and B) identifying the position of the phosphate group of the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate by the elution position of the peak in the MS Is included.
  • the sample is further applied to chromatography.
  • the sample is further applied to ion mobility separation (IMS).
  • the position of the phosphate group of phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate can be specified by comparison with a standard product.
  • the elution position and elution time of a standard product can be calculated and set in advance, and the identification can be performed by comparing with the calculation.
  • a method for identifying PIPs and LPIPs for example, whether stable isotopes of phosphoric acid and inositol ( 13C and deuterium) can be added to a sample in advance can be replaced with natural ones. There is a method to investigate.
  • the present invention provides a method for measuring, detecting or identifying phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate in a sample.
  • the method comprises A) applying the sample to ion mobility separation-mass spectrometry (IMS-MS); and B) depending on the elution position of the peak in the IMS-MS, the phosphorous of the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate.
  • IMS-MS ion mobility separation-mass spectrometry
  • the presence or absence of an acyl group contained in the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate is specified in the specifying step in the present invention.
  • Some phospholipids in the sample do not have an acyl group, but they are not deacylated by the measurement of the present invention, and therefore can be measured in an intact state. Therefore, the presence or absence of an acyl group is also specified. can do.
  • the total molecular weight of acyl groups contained in the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate is also specified in the specifying step in the present invention.
  • the molecular weight is specified by mass spectrometry. Therefore, once it is specified that the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate is specified, the total molecular weight of the acyl group bonded is specified from the molecular weight.
  • the type of acyl group contained in the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate is also specified in the specifying step in the present invention.
  • the molecular weight is specified by mass spectrometry.
  • the total molecular weight of the bound acyl group is determined from its molecular weight, and in the case of phosphatidylinositol phosphate, In order to be specified, the molecular weight of one of the acyl groups is specified. When one molecular weight is specified, the other molecular weight is also specified, and the type of acyl group is specified from the molecular weight.
  • a method for identifying the type of acyl group from the molecular weight based on the results of measurement by MS and MS / MS is described, for example, in Clark J et al. , Nat Methods. 2011 Mar; 8 (3): 267-72. Doi: 10.1038 / nmeth can be referred to.
  • the measurement, detection or identification in the present invention is characterized in that phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate is distinguished from other inositol-containing phospholipids.
  • the measurement, detection or identification method of the present invention can be carried out while retaining the acyl group. More specifically, phosphatidyl phosphate and lysophosphatidylinositol phosphate can be used.
  • inositol-containing phosphoric acid such as glycerophosphoinositol can be distinguished from each other in that the molecular weight of the acyl group is different by measuring the retention of the acyl group.
  • the sample to be measured according to the present invention is prepared under conditions where phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate are not decomposed.
  • the conditions in which phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate are not degraded can include any conditions in which degradation is not performed, and in particular, deacylation treatment is performed. In addition to this, there are other conditions such as not performing a treatment for denaturing the sample and performing a treatment for introducing a protecting group into a phosphate group.
  • liquid column chromatography used in the present invention, a chiral column, a reverse phase column, a normal phase column, an ion exchange column or the like can be used, and any column chromatography which separates a hydrophilic group and a hydrophobic group can be used. .
  • chromatography gas chromatography or liquid column chromatography
  • ion mobility separation can more clearly separate the position of the phosphate group of phosphoinositide and / or lysophosphoinositide.
  • Any chromatography eg, liquid column chromatography
  • ion mobility separation and further mass spectrometry can be used to analyze the phosphate group of phosphoinositide and / or lysophosphoinositide while retaining the acyl group.
  • the position can be separated and measured, detected or identified.
  • the position of the phosphate group of phosphoinositide can be separated and measured, detected or identified while retaining the acyl group by using mass spectrometry (MS), and Furthermore, the position of the phosphate group can be separated and measured, detected or identified in combination with chromatography such as liquid chromatography using a chiral column. Or when using ion mobility separation, you may combine with mass spectrometry as it is, without using chromatography.
  • MS mass spectrometry
  • the method of the invention further comprises the step of quantifying phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate.
  • the sample used in the present invention is an intact sample.
  • that the sample is intact includes conditions under which acyl groups of phosphoinositide and lysophosphoinositide are retained, such as denaturation by heating, oxygen in the air, moisture, heat, light, metal ions, Examples thereof include, but are not limited to, oxidation by the action of microorganisms or enzymes, hydrolysis in an aqueous solvent, deacylation in the presence of an alkylamine such as methylamine, and the like.
  • the position of the phosphate group of phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate is determined without labeling the sample. Since it is not necessary to label with a fluorescent label or radioisotope, it is possible to perform analysis under the condition of the living body as it is or similar conditions to the sample in the living body, reflecting more living body information Analysis can be performed.
  • the present invention provides an apparatus for measuring, detecting or identifying the positions of acyl and phosphate groups of phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate in a sample.
  • the apparatus comprises a mass spectrometer, preferably further comprising a chromatography apparatus or an ion mobility separation apparatus, for example the chromatography apparatus is at least one for gas chromatography and liquid chromatography.
  • One device may be included.
  • the present invention includes a mass spectrometer (MS) apparatus and an application unit that applies the sample to the MS under a condition that the phosphatidylinositol phosphate is not decomposed.
  • MS mass spectrometer
  • the present invention includes a liquid column chromatography-mass spectrometry (LC-MS) apparatus and an application unit that applies the sample to the LC-MS under conditions in which phosphatidylinositol phosphate is not decomposed. Including.
  • LC-MS liquid column chromatography-mass spectrometry
  • the present invention provides an apparatus for measuring, detecting or identifying the positions of acyl groups and phosphate groups of phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate in a sample.
  • the apparatus includes an ion mobility separation-mass spectrometry (IMS-MS) apparatus and an application unit that applies the sample to the IMS-MS under a condition in which phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate is not decomposed.
  • IMS-MS ion mobility separation-mass spectrometry
  • means for performing ion mobility separation may be included as necessary.
  • liquid column chromatography used in the present specification is selected from the group consisting of chiral columns, reverse phase columns, normal phase columns, ion exchange columns, columns that separate hydrophilic groups and hydrophobic groups.
  • it further comprises means for detecting or quantifying the phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate.
  • means for quantifying for example, a method of calculating and quantifying the obtained detection peak in comparison with an internal standard can be mentioned.
  • the present invention provides a method for separating or purifying phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate in a sample depending on the position of a phosphate group while retaining an acyl group.
  • the method comprises A) applying the sample to mass spectrometry (MS); and B) from the eluate of the MS, a phosphatidylinositol phosphate having a phosphate group at the target position identified by the elution position of the peak and / or Or collecting lysophosphatidylinositol phosphate.
  • the sample is further applied to chromatography or ion mobility separation (IMS).
  • the present invention provides a mixture of phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate contained in the mixture from a mixture of two or more of the phosphate groups of phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate.
  • a method for producing a sample comprising phosphatidylinositol phosphates and / or lysophosphatidylinositol phosphates having phosphate groups at fewer (eg, one) positions than the number of acid group positions.
  • the method includes: A) applying the sample to chromatography, ion mobility separation (IMS) or mass spectrometry (MS); and B) identifying the elution from the chromatography, IMS or MS by the elution position of the peak. Collecting a fraction containing a phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate having a phosphate group at a desired target position.
  • the sample is applied to any combination of chromatography, ion mobility separation and mass spectrometry.
  • any technique known in the art may be used as a technique for collecting phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate having a phosphate group at a target position specified by the elution position of the peak.
  • an appropriate device such as an autosampler can be used, or the data can be collected manually.
  • phosphatidylinositol phosphate and / or lysophosphatidylinositol phosphate mixture containing two or more phosphate groups include biological samples, foods, metabolites and any other phosphoinositide and / or lysophosphatidylinositol phosphate. Any sample containing or presumed to contain can be mentioned.
  • the mixture is composed of three isomers of monophosphate phosphatidylinositol (phosphatidylinositol-3-monophosphate, phosphatidylinositol-4-monophosphate, phosphatidylinositol-5-monophosphate).
  • the phosphatidylinositol phosphate having a phosphate group at one position and the lysophosphatidylinositol phosphate having a phosphate group at one position each have a binding pattern of seven kinds of phosphate groups.
  • phosphatidylinositol-3-monophosphate phosphatidylinositol-4-monophosphate, phosphatidylinositol-5-monophosphate, phosphatidylinositol-3,4-diphosphate, phosphatidylinositol- 3,5-diphosphate, phosphatidylinositol-4,5-diphosphate, phosphatidylinositol-3,4,5-triphosphate, and their lysates. Techniques that can be separated in part from the mixture are also the subject of the present invention.
  • the number of phosphatidylinositol phosphates and / or lysophosphatidylinositol phosphates contained in the mixture is less than the number of types of phosphate groups of the lysophosphatidylinositol phosphates” means phosphatidylinositol phosphates and / or lysophosphatidylinositol phosphates contained in the mixture
  • the number of types of the position of the phosphoric acid group is n, it means n-1 or less.
  • the number of phosphatidylinositol phosphates and / or lysophosphatidylinositol phosphates contained in the mixture is less than the number of types of the phosphate groups of the phosphatidylinositol phosphate, but is not limited thereto, but is not limited to one, two, three There may be four types.
  • P phosphatidylinositol PI3P or PI (3)
  • P phosphatidylinositol-3-monophosphate PI4P or PI
  • P phosphatidylinositol-4-monophosphate PI5P or PI
  • P phosphatidylinositol-5-mono Phosphoric acid (PI3P or PI4P or PI5P is expressed as PIP1) PI (3,4)
  • P 2 Phosphatidylinositol-3,4-diphosphate PI (3,5)
  • P 2 Phosphatidylinositol-3,5-diphosphate PI (4,5)
  • P 2 Phosphatidylinositol- 4,5 diphosphate (PI (3,4) P 2 or PI (3,5) P 2 or PI (4,5) P 2 and referred to as PIP2)
  • P 3,4,5) P 3 Phosphatid
  • LPIPs Structures bonded to fatty acids or hydrocarbons through only one of the sn-1 hydroxyl group and sn-2 hydroxyl group of glycerol of PIPs are collectively referred to as LPIPs.
  • Example 1 Production and identification of lysophosphatidylinositol phosphates
  • lysophosphatidylinositol phosphates were produced, and a novel identification method for the new substance was also developed.
  • Phosphatidylinositol phosphates used as starting materials are 17: 0/20: 4-PI3P, 17: 0/20: 4-PI4P, 17: 0/20: 4-PI5P, 17: 0/20: 4-PI ( 3,4) P 2 , 17: 0/20: 4-PI (3,5) P 2 , 17: 0/20: 4-PI (4,5) P 2 , 17: 0/20: 4-PI (3,4,5) P 3 and other glycerophospholipids standard synthetic were purchased from Avanti Polar lipids (Alabaster, AL, USA). Trimethylsilyldiazomethane (TMS-diazomethane) was purchased from Tokyo Kasei. Ultrapure water was obtained from Kanto Chemicals (Tokyo, Japan). All other solvents were HPLC or LC-MS grade and other chemical reagents were analytical grade. These were obtained from Wako Pure Chemicals.
  • TMS-diazomethane Trimethylsilyldiazomethane
  • LPIPs phosphate group methylation protection reaction was performed for highly sensitive measurement with a mass spectrometer. A specimen containing LPIPs was dissolved in 800 ⁇ L of chloroform / methanol (1/1), 150 ⁇ L of trimethylsilyldiazomethane was added, and the mixture was reacted at room temperature for 5 minutes. The reaction was quenched by adding 10 ⁇ L of glacial acetic acid. After adding 800 ⁇ L of methanol / water / chloroform (48: 47: 3) and 400 ⁇ L of chloroform and stirring, the organic layer was dried with N 2 gas. Methanol / 70% ethylamine (1: 0.13%), v / v) 27 ⁇ L and water 9 ⁇ L were added and stirred.
  • LPIPs were measured by the selective reaction monitoring (SRM) method in positive ion mode. Table 2 summarizes the measurement conditions of individual LPIPs in the SRM mode. LPIP1, LPIP2, and LPIP3 were identified by the combination of the m / z value (Q1) of the parent ion and the m / z value (Q2) of monoacylglycerol measured as the daughter ion. A schematic diagram is shown in FIG. 1-1.
  • the abscissa represents the reaction time, and the ordinate represents the production amount of 17: 0 LPIP1 or 20: 4 LPIP1 (the yield obtained from the peak area ratio of the raw material to the reaction product on the chromatogram).
  • LPIP1 was not detected, even when the reaction time was shortened to 60 minutes.
  • LPIP1 was detected only when the reaction time was further reduced to 5, 10, and 30 minutes.
  • Both 17: 0 LPIP1 and 20: 4 LPIP1 were generated.
  • a similar reaction time-dependent tendency was observed in the reaction with a methylamine concentration of 2.14%, which is lower than that of the conventional method. In this case, a small amount of LPIP1 was detected even at reaction times of 60 minutes and 120 minutes, and more LPIP1 was detected at 5, 10, and 30 minutes.
  • the reaction depends on the temperature. Incubation at 10.7% methylamine at 37 ° C. for 10 minutes yielded LPIP1 in a higher yield than the reaction at 53 ° C.
  • LPIPs By adjusting the degree of deacylation by shortening the reaction time, reducing the methylamine concentration, reducing the reaction temperature, and a combination thereof, only one acyl can be deacylated, corresponding to diacyl-type PIPs used as starting materials LPIPs can be synthesized.
  • Table 3 shows examples of LPIPs that can be synthesized using a commercially available PIPs synthetic product as a raw material in this example
  • Table 4 shows an example in which the position of the phosphate group can be determined.
  • the numbers in the table indicate the number of carbon atoms and the number of double bonds.
  • FIG. 2 the results of LPIPs produced from seven kinds of PIPs raw materials (acyl groups are 17: 0, 20: 4) having different phosphorylation patterns are illustrated.
  • the reaction product was dried with N2 gas, the methylation protection of the phosphate group of the target LPIPs was performed, and measurement was performed by the above method using a triple quadrupole mass spectrometer. Using any PIPs as a raw material, the production of LPIPs was observed by the methylamine reaction.
  • the PIPs fraction was added to mobile phase A (methanol / H 2 O / 70% ethylamine (20: 80: 0.13, v / v)) / mobile phase B (methanol / H 2 O / 2-propanol / 70% ethylamine ( 5: 5: 90: 0.13, v / v)) ratio of 90% / 10% (0-0.1 min), with a gradient of 70% / 30% over 0.1-3 min, followed by 10 Separation by a gradient with% / 90% (3-15 min).
  • the flow rate was 30 ⁇ L / min and chromatography was performed at 30 ° C.
  • LPIP1 The identification of LPIP1 will be described with reference to the chromatogram in FIG. 3A and the structural formulas of the starting material, product, and fragment in FIG. 3B.
  • the results of accurate mass spectrometry of LPI (4) P and fragments derived from LPI (4) P are shown as representative examples of LPIP1 synthesis reaction.
  • FIG. 3A the accurate mass measurement results of 17: 0 LPIP 1 (first stage) and its fragment ion (second stage) and 20: 4 LPIP 1 (third stage) and its fragment ion (fourth stage) are determined. Together with the molecular formula.
  • m / z 327.2892 has an error of 0.48 ppm (5 ppm or less) from the theoretical value of m / z determined from the molecular formula of 17: 0 monoacylglycerol (17: 0 MG) (monovalent positive ion with one hydroxyl group missing).
  • m / z 361.22736 has an error of 0.45 ppm (5 ppm or less) from the theoretical value of m / z determined from the molecular formula of 20: 4 monoacylglycerol (20: 4 MG) (monovalent positive ion from which one hydroxyl group is missing).
  • FIG. 3C shows two types of LPIs produced by a mild deacylation reaction of 37: 4 PI (4,5) P2 (sn-1 17: 0, sn-2 20: 4 PI (4,5) P2) ( As a representative example of LPIP2 synthesis reaction, the results of accurate mass spectrometry of fragments derived from LPI (4,5) P2 and LPI (4,5) P2 are shown for 4,5) P2. 17: 0 LPIP 2 (first step) and its fragment ion (second step), 20: 4 LPIP 2 (third step) and its fragment ion (fourth step) accurate mass measurement results together with the determined molecular formula Show.
  • FIG. 3E shows the results of a mild deacylation reaction of 37: 4 PI (3,4,5) P3 (sn-1 17: 0, sn-2 20: 4 PI (3,4,5) P3).
  • the result of carrying out the accurate mass spectrometry of the fragment derived from LPI (3,4,5) P3 and LPI (3,4,5) P3 is shown for the species LPI (3,4,5) P3.
  • the error between these values and the theoretical m / z value obtained from the molecular formulas of 17: 0 LPIP3 (7 methylated product, ethylamine adduct) and 20: 4 LPIP3 (7 methylated product, ethylamine adduct) is 1.
  • 3A to 3F are proof of identification of LPIPs by accurate mass measurement.
  • NMR analysis of LPIPs The structure of the synthesized LPIPs was determined by NMR. Regarding the structure determination of lipids by NMR, those skilled in the art can appropriately determine the structure, for example, 1 H chemical shift is described in Ong et al. Mol. Biosys. (2009) 5, 288-298 can be referred to.
  • the first position gives a chemical shift of about 3.7 ppm, the second position about 5.2 ppm, and the third position about 4.1 ppm, so that it is possible to distinguish between sn-1 and sn-2.
  • aCH 2 gives a chemical shift of about 2.3 ppm and bCH 3 gives a chemical shift of about 1.6 ppm, so that it is possible to distinguish between sn-1 and sn-2.
  • the polyunsaturated portion of the polyunsaturated fatty acid gives a chemical shift of about 2.8 ppm and about 5.3 ppm, it can be used to estimate the type of fatty acid in the lysophospholipid.
  • the phosphorus atom gives a chemical shift of 0 to 2 ppm ( 31 P, about 0.5 ppm low magnetic field shift with lysophospholipidation), and can be used for investigation of reaction progress.
  • the lipid structure can be determined from NMR as shown in FIG. 4A.
  • Example 2 Analysis in biological sample and separation of LPIPs (cultured cells)
  • experiments were conducted on the separation and analysis of LPIPs in biological samples.
  • a human cultured cell line was used as the biological sample.
  • HEK293T human embryo-derived kidney epithelial cell line, Open biosystems catalog number HCL4517
  • Jurkat human acute T-cell leukemia cell-derived cell line, ATCC catalog number TIB-152 under the conditions recommended by the provider Cultured and maintained.
  • Lipid extraction was performed based on the Bligh & Dyer method (EG Bligh et al, Canadian Journal of Biochemistry and Physiology, 1959, 37 (8): 911-917, 10.1139 / o59-099). .
  • FIG. 5A shows the presence of LPIP1, LPIP2, and LPIP3 in HEK293T cells and FIG. 5B in Jurkat cells.
  • the horizontal axis represents molecular species having different fatty acid structures, and the vertical axis represents the abundance in cell-derived phospholipids containing PS 1 nmol.
  • LPIPs having various acyl groups having 16 to 22 carbon atoms and 0 to 6 double bonds were found in cells.
  • LPIP1 and LPIP2 were trace amounts of phospholipids present at a level of 1 / hundred to several tenths of PS, and LPIP3 was present at a level of 1 / hundred to several thousandths.
  • 5K and 5D show the results of detection of 18: 0 LPIP3 and its specific fragments in the analysis of HEK293T cell lipid extract by “Measurement and detection method using hybrid quadrupole-orbitrap mass spectrometer”. Shown in
  • the dehydroxylated product is shown below.
  • the pre-dehydration oxidation is shown below.
  • Example 3 Analysis and separation of LPIPs in biological samples (mouse tissue and human blood samples) Next, in this example, an experiment for separation and analysis of LPIPs in a biological sample was performed using another sample. As biological samples, LPIPs in mouse or human tissues or serum or plasma were used.
  • mice C57BL / 6J ⁇ purchased from Clea Japan Co., Ltd.>, 12-16 weeks old organ / tissue: Brain, heart, liver, large intestine were prepared as follows. Mice were dislocated from the cervical vertebrae, fixed on their back and then laparotomized. Organs isolated after perfusion and blood removal with PBS were immediately frozen in liquid nitrogen. Thawed immediately before lipid extraction and homogenized with a homogenizer. Extraction of phospholipid, separation with DEAE cellulose and methylation reaction were performed according to Example 2. The analysis was also carried out in the same manner as in Example 2, “Measurement and detection method by SRM with triple quadrupole mass spectrometer”.
  • Serum An anesthetic (ketamine) was opened 3 minutes after intraperitoneal administration, and blood was collected from the heart. The collected blood was allowed to stand at room temperature for 15 minutes and then allowed to stand at 4 ° C. for 12 hours. The clot was removed by centrifugation (600 g, 30 minutes), and serum was prepared. Extraction of phospholipid, separation with DEAE cellulose and methylation reaction were performed according to Example 2. The analysis was also carried out in the same manner as in Example 2, “Measurement and detection method by SRM with triple quadrupole mass spectrometer”.
  • Plasma The abdominal cavity was opened 3 minutes after administration of anesthetic (ketamine) and 0.5 ml of blood was collected from the lower abdominal aorta. The collected blood was collected in a 1.5 mL tube containing 2 mg of EDTA-2Na cooled with ice, and a plasma sample was prepared from the supernatant after stirring and centrifugation (600 g, 30 minutes). Extraction of phospholipid, separation with DEAE cellulose and methylation reaction were performed according to Example 2. The analysis was also carried out in the same manner as in Example 2, “Measurement and detection method by SRM with triple quadrupole mass spectrometer”.
  • Human serum Blood was collected from a peripheral vein into a test tube, and the collected blood was allowed to stand at room temperature for 15 minutes, and then allowed to stand at 4 ° C. for 12 hours. The clot was removed by centrifugation (600 g, 30 minutes), and serum was prepared. Extraction of phospholipid, separation with DEAE cellulose and methylation reaction were performed in the same manner as in Example 3. The analysis was carried out in the same manner as in Example 2, “Measurement and detection method by SRM with triple quadrupole mass spectrometer”.
  • LPIPs were present in the mouse tissue. Furthermore, since LPIPs are present as liquid components in the living body such as the serum shown in FIG. 6B and the plasma shown in FIG. It was suggested that the production of LPIP2 by phosphorylation of the protein also occurs outside the cell. LPIPs were also present in human serum as shown in FIG. 6D.
  • Example 4 Use as a cancer marker
  • the usefulness of novel LPIPs was examined.
  • PTEN flox / flox mice subject mice
  • FIG. 7A shows the tissue weight in the upper row and the tissue image by HE staining in the lower row of the prostate of a normal mouse (Ctrl) or a mouse (PTEN KO) that specifically lacks the tumor suppressor gene Pten. It can be seen that Pten deficiency causes prostate cancer.
  • FIG. 7B it was revealed that 16: 0 LPIPs were increased in prostate cancer tissue as compared to the normal prostate as a control. In particular, in LPIP3, a significant increase was found with tumor formation when the normal tissue was below the detection limit. Since the fluctuation of LPIPs correlates with carcinogenesis, the usefulness of LPIPs as biomarkers reflecting cancer treatment targets and cancer is expected.
  • Example 5 Use as an inflammation marker
  • C57BL / 6J purchased from Clea Japan Co., Ltd.>
  • 12-16 week old mice were dissected after dislocation of the cervical vertebrae to obtain the foot femur.
  • Myelospheres and neutrophils were collected from the obtained femur and stimulated with complement component C5a, which is an inflammatory substance.
  • Extraction of phospholipid, separation with DEAE cellulose and methylation reaction were carried out in the same manner as in Example 2.
  • the analysis was also carried out in the same manner as in Example 2, “Measurement and detection method by SRM with triple quadrupole mass spectrometer”.
  • LPIP3 is increased with stimulation of complement component C5a involved in the activation of blood cells related to inflammation such as neutrophils and macrophages.
  • complement component C5a involved in the activation of blood cells related to inflammation such as neutrophils and macrophages.
  • LPIP3 is involved in chemotaxis of inflammatory cells, active oxygen production, phagocytosis, enzyme secretion reaction, etc. is presented, and it can be expected to be useful as a therapeutic target for inflammation.
  • Example 6 Separation of phosphoinositide by column
  • PIPs phosphate group of phosphoinositide
  • Each sample was treated with TMS diazomethane to protect the phosphate groups. Specifically, each sample was treated with 150 ⁇ L of 0.6 M TMS diazomethane (22-25 ° C.), and after 5 minutes, 15 ⁇ L of glacial acetic acid was added to stop the methylation reaction.
  • Mass spectrometer QTRAP6500 with SelexION System (ABSciex, Tokyo, Japan) Pump: Nexera X2 system (Shimadzu Corporation, Kyoto, Japan) Autosampler: PAL HTC-XT (AMR, Tokyo, Japan) Column: CHIRALPAK IC-3, 2.1 mm x 250 mm, particle size 3 ⁇ m (DAICEL corporation, Osaka, Japan) Flow rate: 0.1 mL / min Injection sample volume: 10 ⁇ L (Chromatographic conditions) Mobile phase A: acetonitrile + 5 mM ammonium acetate Mobile phase B: methanol + 5 mM ammonium acetate * All these reagents were LC-MS grades purchased from Wako Pure Chemical (Tokyo, Japan).
  • FIG. 1 shows the results of isomer separation using a 17: 0/20: 4 synthetic product.
  • Phosphoinositides with different numbers of phosphate groups were eluted with different retention times. Even when three types of isomers differing only in the position of the phosphate group were mixed (mixture of PIP or PIP2), they could be separated well by a 20-minute measurement method using a column. As described above, the separation of the phosphoinositide by the phosphate group positional isomer enables regioisomer-specific analysis.
  • This sample was subjected to the following methylation treatment.
  • the sample was treated with 150 ⁇ L of 0.6 M TMS diazomethane (22-25 ° C.), and after 5 minutes, 15 ⁇ L of glacial acetic acid was added to stop the methylation reaction. Thereafter, 700 ⁇ L of a mixed solution of chloroform: methanol: water (3:48:47) was added to each sample, and after centrifugation, the organic layer (lower layer) was dispensed into a Spitz test tube and evaporated with a nitrogen gas concentrator. Each sample was dried and redissolved in 100 ⁇ L of acetonitrile to prepare a sample for LC-MS measurement.
  • FIG. 3 shows the results of 50 repeated measurements (injection sample amount 10 pmol) for each sample in order to investigate the variation between the measurement runs for each phosphoinositide.
  • the dispersion coefficient was 8.4 to 12.0%, indicating that the variation between measurement runs was small.
  • Example 8 Separation of Phosphoinositide by Ion Mobility Separation
  • Example 8 demonstrates that separation by the position of the phosphate group of phosphoinositide by ion mobility separation is possible.
  • the PIP2 isomer was separated by a method other than a chiral column (mass spectrometer only). Attempts were made to separate intact PIP2 (C17: 0 / C20: 4-PI (3,5) P2, -PI (3,4) P2 and -PI (4,5) P2) by using ion mobility. Details are shown below.
  • PI (4,5) P2 could be separated from PI (3,5) P2 and PI (3,4) P2 due to the difference in compensation voltage (COV).
  • COV compensation voltage
  • Example 9 Analysis of evaluation of change in phosphoinositide composition
  • Example 9 shows that changes in phosphoinositide composition in drug-treated cells can be evaluated using the method of the present invention.
  • the elution order of the positional isomers of phosphate groups was confirmed in advance for each diacylglycerol species.
  • HEK293T cells human embryonic kidney epithelial cell line, Open biosystems (Colorado, USA) catalog number HCL4517
  • day 1 ⁇ 10 6 cells / 10 cm dish day 0
  • H 2 O 2 was added to the culture medium to a final concentration of 10 mM, and the cells were scraped and collected with a cell scraper after 0 minutes, 2 minutes, 5 minutes, and 15 minutes, respectively. After centrifuging for 3 minutes, the pellet was recovered, and further washed with PBS ( ⁇ ) to recover the cells.
  • Lipids adsorbed on the column were mixed with chloroform: methanol (1: 1, v / v) (3 mL) and chloroform: methanol: 28% aqueous ammonia: acetic acid (200: 100: 3: 0.9, v / v) ( 3 mL).
  • Highly acidic lipids including PIPs, PI and PS were then eluted with chloroform: methanol: HCl: water (12: 12: 1: 1, v / v) (1.5 mL). After adding 0.75 mL of water and 0.1 mL of 1M NaCl, the solution was shaken, centrifuged and the lower layer was collected.
  • FIG. 13 show an extracted ion chromatogram of 38: 4 phosphoinositide. It is shown that PI (4,5) P2 decreases and PI (3,4) P2 increases as the H 2 O 2 treatment time increases.
  • FIGS. 14A-D show how PIPs with different diacylglycerols change with H 2 O 2 treatment.
  • Statistical processing used one-way analysis of variance and Tukey's multiple comparison test. PIPs with different diacylglycerols were also shown to decrease PI (4,5) P2 and increase PI (3,4) P2 with increasing H 2 O 2 treatment time.
  • Example 10 Evaluation of changes in phosphoinositide composition in genetically engineered mice
  • Example 6 shows that changes in phosphoinositide composition in genetically engineered mice can be evaluated using the method of the present invention.
  • Example 6 the elution order of the positional isomers of phosphate groups was confirmed in advance for each diacylglycerol species.
  • INPP4B has the following dephosphorylation reaction PI (3,4,5) P3 ⁇ PI (3,4) P2 ⁇ PI (3)
  • P PTEN promotes the following dephosphorylation reaction PI (3,4,5) P3 ⁇ PI (4,5) P2 (Kofuji S et. Al., Cancer Discov. 2015 Jul; 5 (7): 730-9, Li Chew C et.al., Cancer Discov. 2015 Jul; 5 (7) : 740-51 and VoTT et.al., Cancer Discov. 2015 Jul; 5 (7): 697-700).
  • mice generated according to previous papers were used in the experiments. Specifically, mice were prepared as follows. A conditional targeting vector was constructed, and the gene fragment containing the 21st coding exon of the mouse Inpp4b gene was deleted by homologous recombination. One loxP site was introduced into intron 20 and two loxP sites were introduced into intron 21. The LacZ-PGK-Neo r cassette was inserted between the two lox sites in intron 21 in the antisense orientation relative to the transcription of Inpp4b.
  • the linear construct was introduced into 1 ⁇ 10 7 E14K mouse embryonic stem (ES) cells by electroporation (Sasaki T et. Al., Science 2000; 287: 1040-6).
  • ES cell colonies resistant to G418 0.3 mg / mL; Life Technologies
  • Recombinant clones were confirmed by standard Southern blots of HindIII digested genomic DNA fragments using a 590 bp probe.
  • Targeted ES cells were injected into C57BL / 6J blastocysts (CLEA Japan, Tokyo, Japan). Chimeric male mice were mated with C57BL / 6JJ female mice to achieve germline inheritance.
  • Inpp4b + / flox mice in which the selection cassette was deleted were produced by mating with MeuCre40 transgenic mice (Leneuve P et. Al., Nucleic Acids Res 2003; 31: e21). Similarly, Inpp4b + / ⁇ mice were produced by crossing Inpp4b + / flox mice with MeuCre40 mice. PCR confirmed that Inpp4b ⁇ / ⁇ mice were different from Inpp4b + / ⁇ mice and Inpp4b + / + mice.
  • An oligo primer common to the Inpp4A + allele and the Inpp4A ⁇ allele (5′-CCTGCCATGGGGTAGTTTTCT-3 ′), a primer specific for the Inpp4A + allele (5′-GTTTACATTTGACAGGGTGGTTGG-3 ′), and the Inpp4A ⁇ allele Primers specific for '-TGCTGTCGCCGAAGAAGTTA-3') were combined in the same PCR reaction.
  • Inpp4b + / ⁇ Pten +/ ⁇ mice were generated by crossing Inpp4b + / ⁇ mice with Pten +/ ⁇ mice (Suzuki A et al., Curr Biol 1998; 8: 1169-78).
  • Inpp4b + / ⁇ Pten +/ ⁇ mice were mated with Inpp4b + / ⁇ mice.
  • Triple mutants were made by crossing Inpp4b + / ⁇ Pten +/ ⁇ mice with Akt1 ⁇ / ⁇ mice or Akt2 ⁇ / ⁇ mice (purchased from Jackson Laboratory). All animal experiments were conducted with the review and approval of the Akita University Animal Experiment Committee.
  • mice prepared in this way the thyroid gland obtained from the following mice was used as a sample.
  • Each sample was subjected to the following lipid extraction and methylation treatment, and measured by chiral LC-MS / MS.
  • each mouse tissue was homogenized with 1.5 mL ice-cold methanol and 50 ⁇ L 0.2 pmol / ⁇ L 17: 0/20: 4-PI3P, 17: 0/20: 4.
  • Lipids adsorbed on the column were mixed with chloroform: methanol (1: 1, v / v) (3 mL) and chloroform: methanol: 28% aqueous ammonia: acetic acid (200: 100: 3: 0.9, v / v) ( 3 mL).
  • Highly acidic lipids including PIPs, PI and PS were then eluted with chloroform: methanol: HCl: water (12: 12: 1: 1, v / v) (1.5 mL). After adding 0.75 mL of water and 0.1 mL of 1M NaCl, the solution was shaken, centrifuged and the lower layer was collected.
  • PI (3,4) P2 was not observed in wild type and INPP4B ( ⁇ / ⁇ ) mice, but production was observed in PTEN (+/ ⁇ ) mice and INPP4B ( ⁇ / ⁇ ) PTEN (+/ ⁇ ) ) Further enhancement of production was observed in mice. This result is consistent with the reported activity of PTEN.
  • FIGS. 16A-D show how PIPs with different diacylglycerols are altered by genetic manipulation.
  • Statistical processing used one-way analysis of variance and Tukey's multiple comparison test. In mice knocked out of a gene that promotes PIP3 degradation, the amount of PIP3 was greatly increased as expected, but a decrease in PIP2 and PIP was not observed.
  • PI (3,4) P2 was not observed in wild type and INPP4B ( ⁇ / ⁇ ) mice, but production was observed in PTEN (+/ ⁇ ) mice and INPP4B ( ⁇ / ⁇ ) PTEN (+/ ⁇ ) ) Further enhancement of production was observed in mice.
  • Example 11 Separation and quantification of phosphate regioisomers of LPIPs
  • the present invention can be used for pharmaceuticals and their development.

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Abstract

L'invention concerne un lysophosphatidylinositol-phosphate, lequel est un nouveau phospholipide, ainsi que les utilisations de celui-ci. L'invention concerne également un procédé de séparation et d'analyse des sites de liaison d'un phospholipide. Plus spécifiquement, le procédé selon l'invention permet de mesurer, détecter ou identifier un phospholipide dans un échantillon, et comporte: (A) une étape dans laquelle une analyse par chromatographie liquide sur colonne - spectrométrie de masse (LC-MS) ou une analyse par spectrométrie de mobilité ionique - spectrométrie de masse (IMS-MS) est appliquée à un échantillon; et (B) une étape dans laquelle en fonction de la position d'élution de pics en LC-MS, la position du groupe phosphate du phospholipide est identifiée.
PCT/JP2017/026563 2016-07-22 2017-07-21 Nouveau phospholipide et ses utilisations ainsi que développement d'un procédé de mesure de séparation de phospholipides Ceased WO2018016645A1 (fr)

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WO2022252443A1 (fr) * 2021-05-31 2022-12-08 上海市食品药品检验研究院 Procédé d'analyse qualitative de composants phospholipidiques dans platycodon grandiflorum et de réalisation de la localisation c = c sur des composants phospholipidiques dans platycodon grandiflorum
CN115684414A (zh) * 2022-11-01 2023-02-03 山西振东制药股份有限公司 盐酸多柔比星脂质体注射液中溶血卵磷脂的检测方法和应用
CN117233281A (zh) * 2023-09-14 2023-12-15 上海奥锐特生物科技有限公司 2-氰乙基-n,n,n’,n’-四异丙基亚磷酰二胺的检测方法

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CN120044110A (zh) * 2023-11-27 2025-05-27 株式会社岛津制作所 确定脂类化学结构的方法以及离子迁移谱串级质谱联用仪

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WO2022252443A1 (fr) * 2021-05-31 2022-12-08 上海市食品药品检验研究院 Procédé d'analyse qualitative de composants phospholipidiques dans platycodon grandiflorum et de réalisation de la localisation c = c sur des composants phospholipidiques dans platycodon grandiflorum
CN115684414A (zh) * 2022-11-01 2023-02-03 山西振东制药股份有限公司 盐酸多柔比星脂质体注射液中溶血卵磷脂的检测方法和应用
CN117233281A (zh) * 2023-09-14 2023-12-15 上海奥锐特生物科技有限公司 2-氰乙基-n,n,n’,n’-四异丙基亚磷酰二胺的检测方法

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