WO2023132338A1 - タンパク質における糖が結合したアミノ酸部位を同定する方法、及びキット - Google Patents

タンパク質における糖が結合したアミノ酸部位を同定する方法、及びキット Download PDF

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WO2023132338A1
WO2023132338A1 PCT/JP2023/000038 JP2023000038W WO2023132338A1 WO 2023132338 A1 WO2023132338 A1 WO 2023132338A1 JP 2023000038 W JP2023000038 W JP 2023000038W WO 2023132338 A1 WO2023132338 A1 WO 2023132338A1
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protein
mass spectrometry
sugar
amino acid
compound
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French (fr)
Japanese (ja)
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俊 松田
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Fujifilm Corp
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Fujifilm Corp
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Priority to EP23737289.1A priority Critical patent/EP4462114A4/en
Priority to CN202380016364.6A priority patent/CN118541607A/zh
Priority to JP2023572478A priority patent/JPWO2023132338A1/ja
Publication of WO2023132338A1 publication Critical patent/WO2023132338A1/ja
Priority to US18/764,888 priority patent/US20240361332A1/en
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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • 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
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/015—Parvoviridae, e.g. feline panleukopenia virus, human Parvovirus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material

Definitions

  • the present invention relates to a method for identifying sugar-bound amino acid sites in proteins.
  • the invention further relates to kits for carrying out the method of identifying amino acid sites.
  • sugar chain modifications to proteins can affect the blood half-life and biological activity. It is necessary. In order to control sugar chain modification to proteins, it is necessary to identify the amino acid sites to which sugars are bound in proteins.
  • Patent Document 1 describes a process of fluorescently labeling a sugar chain in a glycoprotein, a process of obtaining glycopeptide fragments by fragmenting the glycoprotein, and obtaining fluorescence analysis data by fluorescence detection using liquid chromatography. obtaining mass spectrometric data by mass detection using a mass spectrometer; comparing the fluorometric data with the mass spectrometric data to determine the mass-to-charge ratio (m/z) corresponding to the fluorescence intensity peak and extracting the peak of .
  • m/z mass-to-charge ratio
  • the problem to be solved by the present invention is to provide a method that can identify amino acid sites to which sugars are bound in proteins.
  • a further object of the present invention is to provide a kit for carrying out the above-described method for identifying the sugar-bound amino acid site in a protein.
  • the present inventors performed a first mass spectrometry step of mass spectrometry of the fragmented protein, and then bound sugars in the first mass spectrometry step.
  • the protein confirmed to be a It was found that by performing two mass spectrometry steps, it was possible to identify the modified site of GlcNAc in the viral capsid modified with O-glycans.
  • the present invention has been completed based on the above findings.
  • a method for identifying a sugar-bound amino acid site in a protein comprising: a first mass spectrometry step of mass spectrometry of the fragmented protein; A substitution modification step of substituting and/or modifying an amino acid at an amino acid site to which a sugar is bound in the protein confirmed to be sugar-bound in the first mass spectrometry step, and a fragmentation of the protein. process and a second mass spectrometry step of mass spectrometry of the substituted and/or modified and fragmented protein obtained by the above substitution modification step and fragmentation step.
  • ⁇ 2> The method according to ⁇ 1>, comprising comparing the mass spectrometry results obtained from the first mass spectrometry step and the mass spectrometry results obtained from the second mass spectrometry step.
  • ⁇ 3> The method according to ⁇ 1> or ⁇ 2>, wherein the sugar-bound amino acid is serine and/or threonine.
  • ⁇ 4> The method according to any one of ⁇ 1> to ⁇ 3>, wherein the substitution modification step is a substitution reaction by a Michael addition reaction to an amino acid site from which a sugar has been eliminated by a ⁇ -elimination reaction.
  • ⁇ 5> The method according to any one of ⁇ 1> to ⁇ 4>, wherein the Michael addition reaction in the substitution modification step is a reaction with a cyclic active methylene compound.
  • the Michael addition reaction in the substitution modification step is a reaction with a pyrazolone compound, a barbituric acid compound, a dimedone compound, or a hydroxycoumarin compound.
  • the Michael addition reaction in the substitution modification step is a reaction with a pyrazolone compound.
  • ⁇ 8> The method according to ⁇ 7>, wherein the pyrazolone compound is 3-methyl-1-phenyl-pyrazolone.
  • ⁇ 9> The method according to any one of ⁇ 1> to ⁇ 8>, wherein the sugar is N-acetylglucosamine.
  • ⁇ 10> The method according to any one of ⁇ 1> to ⁇ 9>, wherein the protein is a viral capsid.
  • ⁇ 11> The method according to ⁇ 10>, wherein the virus is an adeno-associated virus.
  • ⁇ 12> The method of ⁇ 11>, wherein the virus is adeno-associated virus 5 called AAV5.
  • ⁇ 13> The method according to any one of ⁇ 1> to ⁇ 12>, wherein the amount of protein to be analyzed is 100 fmol or less.
  • FIG. 1 shows MS/MS spectra of O-GlcNAc binding peptides detected from fragmented AAV capsids.
  • FIG. 2 shows MS/MS spectra of O-GlcNAc binding peptides detected from fragmented AAV capsids.
  • FIG. 3 shows the MS/MS spectrum of PMP-ylated and fragmented AAV capsid-derived peptides.
  • FIG. 4 shows the MS/MS spectrum of PMP-ylated and fragmented AAV capsid-derived peptides.
  • FIG. 5 shows an extracted chromatogram of precursor ions for an unmodified peptide preparation and an internal standard (IS).
  • FIG. 6 shows precursor ion extraction chromatograms for S469 modified peptide preparations and an internal standard (IS).
  • FIG. 7 shows the standard curve.
  • AAV means adeno-associated virus.
  • LC means liquid chromatography.
  • MS means mass spectrum.
  • ESI means electrospray ionization.
  • HCD means high energy collisional dissociation.
  • a method for identifying an amino acid site to which a sugar is attached in a protein comprises: a first mass spectrometry step of mass spectrometry of the fragmented protein; A substitution modification step of substituting and/or modifying an amino acid at an amino acid site to which a sugar is bound in the protein confirmed to be sugar-bound in the first mass spectrometry step, and a fragmentation of the protein. process and and a second mass spectrometry step of mass spectrometry of the substituted and/or modified and fragmented protein obtained by the above substitution modification step and fragmentation step.
  • proteins modified with sugars can be identified.
  • the subsequent substitution modification step in the range of amino acid residues found to be modified by sugars in the first mass spectrometry step, the subsequent substitution modification step, fragmentation step and second mass spectrometry step. Since the analysis step identifies the labeled amino acid residue (that is, the amino acid residue modified by sugar), it is possible to identify the site modified by sugar with high accuracy.
  • the sugar-modified site can be identified. .
  • identifying the amino acid site to which the sugar is bound means determining the position of the amino acid to which the sugar is bound.
  • the protein is not particularly limited as long as it is a protein that can be modified with sugar. Proteins may be naturally occurring proteins, proteins obtained by genetic recombination methods, or chemically synthesized proteins. Proteins obtained by genetic recombination include recombinant proteins expressed in host cells.
  • the host cells are preferably eukaryotic cells, more preferably mammalian cells.
  • mammalian cells include, but are not limited to, human cells, mouse cells, rat cells, monkey cells, hamster cells, and the like.
  • human cells can be used.
  • examples of cells include mouse myeloma (NSO) cell line, Chinese hamster ovary (CHO) cell line, HT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BHK (baby hamster kidney cells), VERO, SP2.
  • NSO mouse myeloma
  • CHO Chinese hamster ovary
  • the cells are HEK293 cells, CHO cells, more preferably HEK293 cells.
  • the protein is preferably a viral capsid.
  • the viral capsid also called capsid, is a protein shell that surrounds the viral genome.
  • Viruses include, but are not particularly limited to, adeno-associated viruses, adenoviruses, retroviruses, lentiviruses, Sendai viruses, and the like.
  • the virus is preferably an adeno-associated virus, for example any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11, more preferably AAV5, AAV8 or AAV9. , more preferably AAV5.
  • AAV is produced within the host cell, it is believed that the capsid undergoes sugar modification by host cell-derived enzymes, which may affect the efficacy and side effects of AAV.
  • the FDA (U.S. Food and Drug Administration) guidance published in January 2020 recommends that therapeutic AAVs be analyzed not only for their molecular weight and size, but also for their properties such as sugar modification sites.
  • glycosylation of AAV there is almost no knowledge about glycosylation of AAV, and it is unclear whether AAV is glycosylated or not, and its analysis method has not been established.
  • the methods of the present invention allow analysis of AAV GlcNAc modification sites. This allows the method of the present invention to be used for quality assessment of AAV.
  • the protein may be a protein other than the virus capsid, such as an antibody (e.g., human antibody, humanized antibody, chimeric antibody, mouse antibody, bispecific antibody, etc.), fragmented immunoglobulin, single-chain antibody (scFv). etc. Fragmented immunoglobulins include Fab, F(ab')2, Fv, and the like.
  • the antibody class is not particularly limited, and may be of any class such as IgG such as IgG1, IgG2, IgG3, and IgG4, IgA, IgD, IgE, and IgM, but IgG and IgM are preferred when used as a medicine.
  • Amino acid residues that constitute proteins are not particularly limited, but preferably those containing serine or threonine.
  • the amount of protein to be analyzed may be 1000 fmol or less, 500 fmol or less, 200 fmol or less, or 100 fmol or less.
  • Sugars may be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides (2 to 20 sugar molecules linked together), or polysaccharides (many monosaccharides polymerized). , preferably a monosaccharide.
  • Monosaccharide is a general term for sugars that cannot be further hydrolyzed among sugars.
  • Specific examples of monosaccharides include N-acetylglucosamine (abbreviated as GlcNAc) whose structure is shown below, but are not particularly limited, but N-acetylglucosamine is preferred.
  • amino acid to which sugar is bound is not particularly limited, but is, for example, serine, threonine or asparagine, preferably serine and/or threonine.
  • the sugar (eg, GlcNAc) modification rate in a protein represents the ratio of modified proteins among the protein molecules contained in the sample. number of protein molecules)/(number of protein molecules contained in the sample).
  • the sugar modification rate in the protein may be, for example, less than 1%, preferably 0.8% or less, 0.6% or less, 0.5% or less, or 0.4% or less.
  • the quantification of the sugar modification rate in proteins can be performed as described in Examples below.
  • amino acid sites to which sugar chains are bound can be identified in a protein having a sugar modification rate of 0.0001% or more and less than 1% and 1 fmol or more and 1000 fmol or less. Furthermore, in a protein with a sugar modification rate of 0.001% or more and 0.6% or less and 5 fmol or more and 500 fmol or less, the amino acid site to which the sugar chain binds can be identified. Furthermore, in a protein with a sugar modification rate of 0.005% or more and 0.4% or less and 10 fmol or more and 100 fmol or less, amino acid sites to which sugar chains are bound can be identified.
  • substitution-modification step for proteins confirmed to have sugar-bonded sugars in the first mass spectrometry step, amino acids at sugar-bonded amino acid sites in the protein are substituted and/or modified.
  • the substitution modification step is preferably a Michael addition reaction to the amino acid site from which the sugar has been eliminated by the ⁇ -elimination reaction.
  • the ⁇ -elimination reaction and the Michael addition reaction can be carried out in the same step, where the reaction apparently replaces the substituent on the same atom of the compound.
  • the Michael addition reaction in the substitution modification step is preferably a reaction with a cyclic active methylene compound, more preferably a pyrazolone compound, a barbituric acid compound, a dimedone compound, or a hydroxycoumarin compound, and particularly preferably a pyrazolone compound. It is a reaction by
  • the pyrazolone compound is not particularly limited as long as it is a compound having a pyrazolone group.
  • p-tolyl-5-pyrazolone MTP
  • 3-methyl-1-(quinolin-8-yl)-1H-pyrazol-5(4H)-one Two or more types may be used in combination.
  • the pyrazolone compound is particularly preferably 3-methyl-1-phenyl-pyrazolone (PMP).
  • the concentration of the cyclic active methylene compound in the Michael addition reaction in the substitution modification step is not particularly limited, but the concentration in the reaction solution is generally 0.05 mol/L to 1 mol/L, preferably 0.1 mol. /L to 0.5 mol/L.
  • a compound represented by the following formula (1) can be used as the cyclic active methylene compound.
  • a cyclic active methylene compound may be used in combination of two or more compounds.
  • W and X each represent a substituent containing an electron-withdrawing group, and W and X are linked to each other to form a 5- or 6-membered ring.
  • Electron-withdrawing groups contained in W and X include those having a carbonyl bond or an azomethine bond.
  • Representative active methylene compounds include a pyrazolone compound represented by formula (2), a barbiturate compound represented by formula (3), a dimedone compound represented by formula (4), and a compound represented by formula (5). and hydroxycoumarin compounds represented.
  • Ar represents an aromatic ring optionally having one or more substituents.
  • Aromatic rings include benzene ring, pyrimidine ring, pyridazine ring, pyrazine ring, pyridine ring and the like.
  • substituents on these aromatic rings include alkyl groups having 1 to 8 carbon atoms, alkoxy groups having 1 to 8 carbon atoms, monoalkylamino groups having 1 to 8 carbon atoms, dialkylamino groups having 2 to 16 carbon atoms, and halogen atoms. and a cyano group.
  • R 1 represents an alkyl group having 1 to 8 carbon atoms.
  • R 2 and R 3 are each independently a hydrogen atom, an alkyl group having 1 to 8 carbon atoms, an alkoxycarbonylalkyl group having 3 to 16 carbon atoms, a carbamoylalkyl group having 2 to 9 carbon atoms, or It represents an aromatic ring which may have a substituent.
  • Aromatic rings include benzene ring, pyrimidine ring, pyridazine ring, pyrazine ring, pyridine ring, pyrrole ring, pyrazole ring, imidazole ring, triazole ring and tetrazole ring.
  • substituents on these aromatic rings include alkyl groups having 1 to 8 carbon atoms, alkoxy groups having 1 to 8 carbon atoms, monoalkylamino groups having 1 to 8 carbon atoms, dialkylamino groups having 2 to 16 carbon atoms, and halogen atoms. , and a cyano group.
  • R 4 and R 5 each independently represent an alkyl group having 1 to 4 carbon atoms.
  • R 6 and R 7 are each independently a hydrogen atom, an alkyl group having 1 to 8 carbon atoms, an alkoxy group having 1 to 8 carbon atoms, a monoalkylamino group having 1 to 8 carbon atoms, or a dialkylamino group having 2 to 16 carbon atoms. group, a halogen atom, a cyano group, or the like.
  • the fragmentation step of fragmenting a protein can be performed by allowing a proteolytic enzyme to act on the protein. Pretreatment may include reduction and carbamidomethylation of the protein, followed by protein fragmentation.
  • Pretreatment may include reduction and carbamidomethylation of the protein, followed by protein fragmentation.
  • a cysteine residue in a protein can be reduced by allowing a reducing agent such as dithiothreitol to act on the protein.
  • the reduced protein can then be carbamidomethylated by adding iodoacetamide.
  • the protein can then be fragmented by allowing a proteolytic enzyme to act on the protein.
  • Enzymes that fragment proteins are preferably capable of cleaving proteins between 5 and 100 residues, more preferably between 6 and 60 residues, more preferably 7 residues.
  • proteolytic enzymes include trypsin, chymotrypsin, pepsin, papain, ficin, bromelain, Lys-C (Promega), Asp-N (Promega), Arg-C (Promega), proteinase K, lysyl endopeptidase, V8. proteases, etc., preferably trypsin.
  • the amount of proteolytic enzyme used is preferably 1/20 to 1/100 times the weight of the protein in the sample.
  • the order of the substitution modification step and the fragmentation step of fragmenting the protein is not particularly limited. That is, the fragmentation step of fragmenting the protein may be performed after performing the substitution modification step, or the fragmentation step of fragmenting the protein may be performed before the substitution modification step.
  • the first mass spectrometry step in the present invention is a step of mass spectrometry of the fragmented protein, confirming that the protein is modified by sugar, and furthermore, the range of amino acid residues that have been modified by sugar It is a process for specifying.
  • the amino acid at the amino acid site where the sugar is bound in the protein is substituted and / or modified. and a step of analyzing the protein obtained by carrying out a substitution modification step and a fragmentation step of fragmenting the protein.
  • the method of mass spectrometry is not particularly limited, but as an example, the mass of ions and molecules can be measured by ionizing molecules and measuring their mass-to-charge ratio (m/z).
  • Pretreatment preferably includes reduction, carbamidomethylation and fragmentation of the protein.
  • a cysteine residue in a protein can be reduced by allowing a reducing agent such as dithiothreitol to act on the protein.
  • the reduced protein can then be carbamidomethylated by adding iodoacetamide.
  • the protein can be fragmented by allowing a proteolytic enzyme to act on the protein. Enzymes that fragment proteins are preferably capable of cleaving proteins between 5 and 100 residues, more preferably between 6 and 60 residues, more preferably 7 residues. Particularly preferred is one that cuts between more than 40 residues and less.
  • proteolytic enzymes examples include trypsin, chymotrypsin, pepsin, papain, ficin, bromelain, Lys-C, Asp-N, Arg-C, proteinase K, lysylendopeptidase, V8 and the like, preferably trypsin.
  • trypsin a proteolytic enzyme
  • chymotrypsin pepsin
  • papain ficin
  • bromelain Lys-C
  • Asp-N Asp-N
  • Arg-C proteinase K
  • lysylendopeptidase V8 and the like
  • trypsin preferably trypsin.
  • the fragmented protein (peptide) may be desalted using GL-Tip SDB (GL Sciences) or the like.
  • the protein was subjected to the substitution modification step and the fragmentation step (the order of the substitution modification step and the fragmentation step does not matter).
  • mass spectrometry can be performed.
  • the fragmented protein may be desalted using GL-Tip SDB (GL Sciences) or the like.
  • Mass spectrometric analysis of pretreated samples can be performed using liquid chromatography (LC) and mass spectrometry (MS).
  • the liquid chromatography device and mass spectrometer may be connected in series with each other, or only the mass spectrometer may be used.
  • an LC-MS system configured by serially connecting a liquid chromatography device and a mass spectrometer can be used.
  • tandem-type LC-MS/MS, LC-MS/MS/MS, or the like can be used.
  • the liquid chromatography (LC) device is not particularly limited as long as it can separate sugar-modified proteins by liquid chromatography.
  • liquid chromatography for example, high performance liquid chromatography (HPLC), ultra high performance high separation liquid chromatography (UHPLC, UPLC, or UFLC), or low flow rate LC can be used. equipment can be selected.
  • HPLC high performance liquid chromatography
  • UHPLC ultra high performance high separation liquid chromatography
  • UFLC ultra high performance high separation liquid chromatography
  • low flow rate LC can be used. equipment can be selected.
  • liquid chromatography (LC) low flow LC is preferred.
  • examples of low-flow LC include nanoflow liquid chromatography (nano-LC), capillary LC, micro-LC, etc. Among them, nano-LC is particularly preferred.
  • An LC apparatus generally includes a separation column and a pump that feeds a separation solution into the separation column.
  • the LC apparatus may include other elements than those described above, such as autosamplers, heaters, detectors to detect separated components, and the like.
  • Detectors include, for example, UV detectors and fluorescence detectors.
  • a detector can be connected between the column and the ion source (ionization section).
  • the conditions of the analytical column used for liquid chromatography are not particularly limited and can be appropriately selected.
  • a reversed-phase column can be used as the separation column. Examples of reversed-phase columns include columns packed with octadecylsilyl silica gel packing material and columns with ion-exchange resin mixed therein. Columns used in reversed phase chromatography packed with chemically bound packing (ODS columns) are preferred.
  • ODS columns chemically bound packing
  • As the separation column for example, MonoCap C18 HighResolution 750 (GL Sciences) can be used.
  • a mobile phase concentration gradient can be formed by mixing two or more solutions with different compositions while changing the mixing ratio. Combinations of solutions can be selected as appropriate to form the desired gradient.
  • the flow rate can be appropriately selected according to various conditions such as the inner diameter of the separation column.
  • the flow rate of the separation solution may or may not be constant throughout the separation process. For example, it can be appropriately selected in the range of 100 nL/min to 100 ⁇ L/min.
  • a person skilled in the art can appropriately select the column temperature in liquid chromatography. For example, it is 20 to 70°C, preferably 25 to 50°C.
  • the liquid chromatography/tandem mass spectrometry fragment ion analysis method is an apparatus consisting of a liquid chromatography section and a mass spectrometer section, and the mass spectrometer section further has a section capable of decomposing and detecting precursor ions.
  • a normal mass spectrometer can be used as the mass spectrometer unit. There may be one mass spectrometer, two or more. Two or more mass spectrometers can be used in series. That is, the LC-MS system may be LC-MS/MS or LC-MS/MS/MS.
  • the tandem mass spectrometer can use a magnetic deflection type, a quadrupole type, an ion trap type, a time-of-flight type, an orbitrap type, or a hybrid type thereof.
  • a quadrupole/orbitrap tandem mass spectrometer is preferred.
  • ionization methods in mass spectrometers include electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI), electron ionization (EI), chemical Ionization (CI: Chemical Ionization) method, Field Desorption (FD) method, Fast Atom Bombardment (FAB) method, Atmospheric pressure chemical ionization (APCI: Atmospheric Pressure Chemcal Ionization) on,) method, or inductive coupling
  • ESI electrospray ionization
  • MALDI matrix-assisted laser desorption ionization
  • EI electron ionization
  • CI chemical Ionization
  • FD Field Desorption
  • FAB Fast Atom Bombardment
  • APCI Atmospheric pressure chemical ionization
  • ICP Inmospheric Pressure Chemcal Ionization
  • a quadrupole/orbitrap tandem mass spectrometer is a mass spectrometer consisting of an ion source, a quadrupole (Q1), a collision cell (Q2), an orbitrap (Q3), and a detector.
  • the analyte is ionized in the ion source to produce precursor ions, which are separated by mass in the quadrupole (Q1) according to their mass-to-charge ratio (m/z).
  • product ions are generated by colliding with an inert gas such as nitrogen or argon in the collision cell (Q2) (collision-induced dissociation: CID).
  • CID collision-induced dissociation
  • the spectrum of the mass-to-charge ratio (m/z) of the precursor ion of the peptide and the mass-to-charge ratio of the product ion indicates that the peptide has undergone sugar modification. It can be made clear that Within the range of peptide amino acid residues found to undergo glycosylation, the subsequent substitution modification step, fragmentation step and second mass spectrometry step result in the mass-to-charge ratio (m/ z) and labeled amino acid residues (ie, sugar-modified amino acid residues) can be identified from the mass-to-charge ratio spectrum of the product ions.
  • the sugar chain modification rate can be quantified based on the results of mass spectrometry.
  • the sugar chain modification rate can be quantified, for example, as follows.
  • the modification rate is obtained by quantifying the number of molecules of peptides to be measured (unmodified peptides and GlcNAc-modified peptides having the same amino acid sequence) present in the sample.
  • peptides to be measured and preparations of their stable isotopes are used.
  • Stable isotopes are used as internal standards (IS) for measurements.
  • a calibration curve is obtained using a known amount of peptide preparation to which an internal standard has been added, and this is used to quantify each peptide to be measured in the sample.
  • Quantitation based on the results of mass spectrometry is determined from the ratio of the peak area obtained from the extracted chromatogram of precursor ions corresponding to each peptide to be measured and the peak area obtained from the extracted chromatogram of precursor ions corresponding to each IS. .
  • kits for carrying out the above-described method for identifying sugar-bonded amino acid sites in a protein according to the present invention comprising a cyclic active methylene compound.
  • cyclic active methylene compounds are as described herein above.
  • the kit of the present invention may further contain means for purifying the fragmented protein.
  • means for purifying the fragmented protein include solid-phase extraction using hydrophobic interaction (a solid phase packed with a filler to which ODS groups are chemically bonded).
  • a kit of the invention may comprise a written protocol for carrying out a method of identifying sugar-linked amino acid sites in a protein according to the invention. This protocol includes, for example, identification procedures and information required for identification.
  • the kit of the present invention may further contain reagents used in the above methods. Examples of reagents include proteolytic enzymes and buffers.
  • the AAV pellet was dissolved in 20 ⁇ L of MPEX PTS Reagent B (GL Sciences), dithiothreitol (DTT) with a final concentration of 5 mmol/L was added, and the mixture was allowed to stand at room temperature for 30 minutes to reduce the cysteine residues in the protein. After that, iodoacetamide (IAA) with a final concentration of 25 mmol/L was added, and the mixture was allowed to stand at room temperature for 30 minutes in the dark for carbamidomethylation.
  • DTT dithiothreitol
  • IAA iodoacetamide
  • the flow rate was 500 nL/min, mobile phase A was 0.1% formic acid (in H 2 O) and mobile phase B was acetonitrile.
  • the column temperature was set at 35° C. and the injection volume was 10 ⁇ L.
  • ESI-MS (Positive mode) was used for detection, and the capillary voltage was set to 2 kV.
  • the MS scan range was m/z 350-1800 (resolution 70,000, AGC 3 ⁇ 10 6 ) and the top 10 precursor ions in each MS scan were fragmented with HCD followed by MS/MS scans (resolution 35,000, AGC 1 ⁇ 10 5 ).
  • the normalized collision energy was set to 25% and the dynamic exclusion time was 30 seconds. Isolation width was set to 2.0 m/z.
  • a database search was used for peptide qualitative screening.
  • Proteome Discoverer Ver1.3 (Thermo Fisher Scientific) was used for database analysis, and Sequest HT (Thermo Fisher Scientific) and Mascot (Matrix Science) were used as database search engines. Allowable trypsin uncleavage number within 2, allowable precursor mass error of 10 ppm, fragment mass error of 20 mmu, fixed modification set to carbamidomethylation (C), dynamic modification set to oxidation (O), and HexNAc (S and T) bottom.
  • AAV9 Q6JC40
  • Q6JC40 UniProt accession number Q6JC40
  • the false identification rate was calculated based on the decoy database (reverse amino acid sequence of the database) and set to less than 1% at the peptide level.
  • the MS/MS spectrum of each peptide hit by the search was manually checked, and those with a mass error of 10 ppm or less from the theoretical value of fragment ions were adopted as the basis for qualitative analysis.
  • the glycopeptide candidates hit by the search those in which sugar-derived ion groups (m/z 204 and two or more other fragment ions) were observed in the MS/MS spectrum were judged to be glycopeptides.
  • FIGS. 1 and 2 MS/MS spectra of O-GlcNAc-bound peptides detected from AAV are shown in FIGS. 1 and 2.
  • FIG. FSVAGPSNMAVQGR shown in Figure 1 shows the amino acid sequence of amino acid positions 463-476 of AAV.
  • VSTTVTQNNNSEFAWPGASSWALNGR shown in FIG. 2 indicates the amino acid sequence of amino acid positions 489-514 of AAV.
  • the results in FIG. 1 show that one O-GlcNAc binds to amino acid positions 463-476 of AAV9.
  • the results in FIG. 2 indicate that one O-GlcNAc binds to amino acid positions 489-514 of AAV9.
  • FIGS. 1 shows the amino acid sequence of amino acid positions 463-476 of AAV.
  • the air-dried AAV pellet was dissolved in 20 ⁇ L of MPEX PTS Reagent B, and subjected to reduction by DTT, alkylation by IAA, and fragmentation by trypsin digestion (fragmentation step) in the same manner as in (1) above.
  • Peptide desalting was also performed using GL-Tip SDB in the same manner as in (1) above.
  • FIGS. 3 and 4 MS/MS spectra of PMP-derived AAV-derived peptides are shown in FIGS. 3 and 4.
  • FIG. 3 the difference in the mass-to-charge ratios of the y 8 + and y 7 + ions of the peptide closely matches the mass of the PMP-attached serine, indicating that It was found that serine (S469 of AAV9) was PMPylated.
  • FIG. 4 the difference in the mass-to-charge ratios of the y 8 + and y 7 + ions of the peptide closely matches the mass of the PMP-attached serine, indicating that It was found that serine (S507 of AAV9) was PMPylated. That is, binding of O-GlcNAc to S469 and S507 of AAV9 could be detected.
  • the ion extraction conditions used for peptide quantification are as follows. FSVAGPSNMAVQGR (m/z 710.8559) FSVAGP(GlcNAc-S)NMAVQGR (m/z 715.8578) [ 13C5,15N5 ] FSVAGPSNMAVQGR (m/z 812.3959 ) [ 13 C 5 , 15 N 5 ]FSVAGP(GlcNAc-S) NMAVQGR (m/z 817.3976)
  • Precursor ion extraction chromatograms for the unmodified peptide preparation and the internal standard (IS) are shown in FIG.
  • Precursor ion extraction chromatograms for the S469-modified peptide preparation and the internal standard (IS) are shown in FIG.
  • the peak area of each peptide was calculated from the extracted chromatogram of precursor ions corresponding to each peptide.
  • Each peptide was quantified by the peak area ratio of the target peptides (unmodified and GlcNAc-modified peptides) and their corresponding IS.
  • Each peptide standard product to which IS was added was used to create a calibration curve. The obtained calibration curve is shown in FIG.
  • Modification rate number of GlcNAc-modified peptide molecules/(number of unmodified peptide molecules + number of GlcNAc-modified peptide molecules) Based on , the percent modification was calculated in moles. The modification rate was 0.4%.

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JUN-ICHI FURUKAWA, NAOKI FUJITANI, KAYO ARAKI, YASUHIRO TAKEGAWA, KOTA KODAMA, YASURO SHINOHARA: "A Versatile Method for Analysis of Serine/Threonine Posttranslational Modifications by B-Elimination in the Presence of Pyrazolone Analogues", ANALYTICAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, vol. 83, no. 23, 1 December 2011 (2011-12-01), pages 9060 - 9067, XP055134989, ISSN: 00032700, DOI: 10.1021/ac2019848 *
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