WO2012121302A1 - 分析方法 - Google Patents
分析方法 Download PDFInfo
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- WO2012121302A1 WO2012121302A1 PCT/JP2012/055873 JP2012055873W WO2012121302A1 WO 2012121302 A1 WO2012121302 A1 WO 2012121302A1 JP 2012055873 W JP2012055873 W JP 2012055873W WO 2012121302 A1 WO2012121302 A1 WO 2012121302A1
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- secretin family
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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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/605—Glucagons
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- 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
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- 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
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
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- 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
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
Definitions
- the present invention relates to a method for analyzing glucagon / secretin family peptides and a method for quantifying glucagon / secretin family peptides in a sample using the analysis method.
- the glucagon / secretin family peptide is a generic term for peptides having high amino acid sequence homology with glucagon and the like. Accurately grasping the concentration of glucagon / secretin family peptides in vivo is necessary for functional research, for understanding the disease state of patients, or for developing pharmaceuticals for improving these diseases. is important.
- a quantification method using an antibody is generally used, as represented by enzyme immunoassay (ELISA), radioimmunoassay (RIA) and the like.
- ELISA enzyme immunoassay
- RIA radioimmunoassay
- Non-Patent Documents 1 to 3 Patent Document 1
- Non-Patent Documents 1 and 2 use an antibody as a pretreatment
- Patent Document 1 and Non-Patent Document 3 are not yet satisfactory in terms of quantitative sensitivity and the like.
- Quantitative methods using mass spectrometry are still unsatisfactory in terms of quantitative sensitivity, and the current situation is that establishment of a quantitative method using a small amount of sample is required. Furthermore, if a sample is prepared for mass spectrometry and stored for several days before measurement, the measured value may change.
- the present invention has been made in view of the above, and provides a method capable of stably analyzing a glucagon / secretin family peptide under practical conditions in which a measurement sample is not easily subjected to chemical changes during storage. The purpose is to do.
- the present inventor has noticed that the measurement value of mass spectrometry may or may not be stable depending on the peptide fragment to be measured. And when analyzing a glucagon secretin family peptide, it discovered that selection of the peptide fragment to analyze was important. That is, this analysis method is characterized mainly by cleaving the peptide bond of aspartic acid in the glucagon / secretin family peptide and selecting the peptide fragment on the N-terminal side of the glucagon / secretin family peptide as the measurement target.
- the main configuration of the present invention is as follows. (1) a cleavage step of cleaving the peptide bond of aspartic acid present in the amino acid sequence of the glucagon / secretin family peptide contained in the sample to make the glucagon / secretin family peptide a peptide fragment; the peptide cleaved in the cleavage step Separation and purification step of separating and purifying the fragments by liquid chromatography and selecting a peptide fragment on the N-terminal side of the glucagon / secretin family peptide to be measured; mass spectrometry of the separated and purified peptide fragments, and glucagon / secretin family An analysis step of detecting a peptide fragment on the N-terminal side of the peptide; and a method for analyzing the glucagon / secretin family peptide contained in the sample.
- the glucagon / secretin family peptide is selected from the group consisting of a glucose-dependent insulinotropic polypeptide, glucagon-like peptide-1, glucagon-like peptide-2, glucagon, and analogs thereof (1)
- (6) The analysis method according to any one of (1) to (5), wherein in the separation and purification step, a substance to be measured is selected by mass selection of precursor ions.
- the internal standard substance is a peptide in which one or more amino acids selected from positions 1 to 15 in the amino acid sequence of the glucagon / secretin family peptide are substituted with an amino acid labeled with a stable isotope, (12) or the quantification method according to (13).
- (15) (a) Control matrix, (b) Internal standard substance or solution thereof, (c) Glucagon / secretin family peptide or their standard substance or solution thereof, (d) Cleavage substance, (e) Solid phase A kit for quantification of glucagon / secretin family peptides, including an extraction plate.
- the glucagon / secretin family peptide is selected from the group consisting of a glucose-dependent insulinotropic polypeptide, glucagon-like peptide-1, glucagon-like peptide-2, glucagon, and analogs thereof. Or the quantification method as described in (16).
- the glucagon / secretin family peptides can be analyzed with high accuracy even after a long time after preparation.
- a trace amount of glucagon / secretin family peptide in a sample can be quantified with high sensitivity, and the reproducibility of the quantitative value is good.
- FIG. 3 is a diagram showing a chromatogram when GIP is cleaved with 2% formic acid.
- FIG. 3 is a diagram showing a chromatogram when GIP is cleaved with 2% acetic acid.
- FIG. 3 is a diagram showing a chromatogram when GIP is cleaved with 1% trifluoroacetic acid. It is a figure which shows the chromatogram at the time of cut
- the glucagon / secretin family peptides analyzed in the present invention are peptides having high amino acid sequence homology with glucagon and the like.
- the glucagon / secretin family peptides defined herein include their analogs.
- Examples of the glucagon / secretin family peptides include glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), glucagon and the like. .
- GIP and GLP-1 are known as incretins. This includes those inherent in the living body, those artificially synthesized, and species variants.
- An analog of a glucagon / secretin family peptide is one in which one or more amino acids have been deleted, substituted or added (for example, inserted) in the amino acid sequence of the glucagon / secretin family peptide, Those having substantially the same activity.
- the amino acid sequence of the glucagon / secretin family peptide analog preferably has 80% or more homology with the amino acid sequence of the glucagon / secretin family peptide.
- Such a peptide design is made for the purpose of enhancing the effect, enhancing the selectivity, or stabilizing against peptide degradation, and varies depending on the type of glucagon / secretin family peptide, but can be performed by a method well known to those skilled in the art. it can.
- glucagon / secretin family peptide in which sugar chains, fatty acids, lipids, nucleic acids and the like are bound to peptide chains. That is, the analogs of glucagon / secretin family peptides include derivatives of glucagon / secretin family peptides, as well as modified, chimeric and hybrid forms.
- GIP glucose-dependent insulinotropic polypeptide
- active GIP consisting of 42 amino acids, and is usually expressed as GIP 1-42 [SEQ ID NO: 13].
- the active GIP is cleaved by two residues at the N-terminal amino acid by DPP-IV to become an inactive GIP 3-42 [SEQ ID NO: 14]. Therefore, by measuring the peptide fragment on the N-terminal side of GIP, active GIP and inactive GIP can be distinguished and simultaneously analyzed or quantified.
- one or more amino acids are substituted due to species differences such as human, mouse, or rat.
- Human active GIP is a peptide consisting of the following sequence, but in mouse and rat, His at position 18 is substituted with Arg.
- Non-active GIP analogs include peptides in which the amino acid side of the N-terminal side of these GIP analogs has been cleaved by 2 residues (the amino acid sequence has not changed, and some modified amino acids have The sequence number is omitted).
- GLP-1 Glucagon-like peptide-1
- GLP-1 is usually active as an amide of GLP-1 (7-36) [SEQ ID NO: 25] or GLP-1 (7 -37)
- the amide of [SEQ ID NO: 26] is known.
- International Patent Application No. 91/11457 reports that GLP-1 (7-34) [SEQ ID NO: 27] and GLP-1 (7-35) [SEQ ID NO: 28] are also active.
- the N-terminus of the GLP-1 precursor is expressed as the 1-position, the active GLP-1 has the N-terminus at the 7-position.
- GLP-1 (7-37) is a peptide consisting of the following sequences.
- GLP-1 GLP-1 -active GLP-1 analogs
- non-active GLP-1 analogs include peptides in which two amino acid residues at the N-terminal side of these GLP-1 analogs are cleaved.
- Ser8] -GLP-1 (7-37) SEQ ID NO: 31
- [Gly8] -GLP-1 (7-37) SEQ ID NO: 32
- Exendin is known as a GLP-1 receptor agonist and is also included in the GLP-1 analog.
- Exendin includes Exendin-3 [SEQ ID NO: 56], Exendin-3 amide [C-terminal amide derivative of the same sequence as SEQ ID NO: 56], Exendin-4 [SEQ ID NO: 57], Exendin-4 amide [SEQ ID NO: 57 Cend amide derivative of the same sequence], Exendin-4 acid [acid variant of the same sequence as SEQ ID NO: 57], Exendin-4-LysLysLysLysLys [SEQ ID NO: 58], Exendin-4-LysLysLysLysLysLys [SEQ ID NO: 59], Exendin-4 (1-30) [SEQ ID NO: 60], amide of Exendin-4 (1-30) [C-terminal amide derivative of the same sequence as SEQ ID NO: 60], Exendin-4 (1-28) [SEQ ID NO: 61 ] Exendin
- GLP-2 Glucagon-like peptide-2
- GLP-2 is a 33 amino acid enterotrophic peptide hormone produced through post-translational processing of proglucagon. It is usually written as [SEQ ID NO: 64]. Similar to GIP, DPP-IV cleaves two residues at the N-terminal amino acid and loses its activity, resulting in inactive GLP-2 (3-33) [SEQ ID NO: 65]. GLP-2 can also be analyzed or quantified at the same time by distinguishing active GLP-2 and non-active GLP-2 by measuring the N-terminal fragment. Specifically, the following are well known as analogs of GLP-2.
- Glucagon is a peptide hormone consisting of 29 amino acids, and is usually expressed as Glucagon [SEQ ID NO: 69]. Specifically, the following are well known as analogs of Glucagon.
- Samples include biological samples such as blood, serum, plasma, urine, saliva, exudate, and tissue extract, and in vitro samples such as drugs and cell culture fluid.
- the glucagon / secretin family peptide can be quantified using a sample in a concentration range of 5 ⁇ L to 5 mL and 0.1 pM to 500 pM. More preferably, the above-mentioned quantification can be performed with a sample in a concentration range of 10 ⁇ L to 3 mL and 0.5 pM to 200 pM. If the amount can be quantified with a sample of 5 mL or less, the amount of blood collected can be reduced, so that the burden on patients can be reduced and the extraction operation is not complicated.
- the above-mentioned quantification can be performed with plasma in a concentration range of preferably 5 ⁇ L to 500 ⁇ L and 0.1 pM to 500 pM. More preferably, the above quantification can be performed with plasma in a concentration range of 10 ⁇ L to 300 ⁇ L and 0.5 pM to 200 pM.
- the peptide fragment of the glucagon / secretin family peptide is a peptide obtained by cleaving the glucagon / secretin family peptide by any method, and refers to a continuous part of the amino acid sequence of the glucagon / secretin family peptide.
- the peptide bond of aspartic acid (Asp) in the amino acid sequence of the glucagon / secretin family peptide is cleaved, and the peptide fragment on the N-terminal side of the glucagon / secretin family peptide is mass analyzed. For this cleavage, the N-terminal side of aspartic acid or the C-terminal side may be cleaved.
- Such a peptide fragment is easy to obtain a stable measurement value, and the sensitivity is improved. Further, analysis using mass spectrometry can be performed without using an antibody. Furthermore, it is difficult to generate multivalent ions, and the effect of species difference can be reduced.
- the amino acid sequences from position 1 to position 17 of GIP are highly homologous and are consistent in humans, rats, and mice. For storage stability, it is important that the peptide fragment to be measured does not contain an easily oxidized amino acid. Amino acids that are easily oxidized include methionine (Met), tryptophan (Trp), and cysteine (Cys).
- the peptide fragment on the N-terminal side of the glucagon / secretin family peptide does not contain the amino acid and has good storage stability. If the peptides are different glucagon / secretin family peptides, the peptide fragments in this method will be different. It seems that there is no peptide having the same sequence as the N-terminal peptide cleaved with aspartic acid of the glucagon / secretin family peptide in the living body. From the above two points, the peptide fragment on the N-terminal side of the glucagon / secretin family peptide is distinguished by mass spectrometry and can be analyzed with high specificity.
- the peptide bond of aspartic acid in the amino acid sequence can be cleaved using a substance that cleaves the peptide bond (cleaving substance).
- a substance that cleaves a peptide bond include sequence-specific cleavage protease or acid.
- An example of a sequence-specific cleavage protease is endopeptidase Asp-N. Endopeptidase Asp-N hydrolyzes the peptide bond on the N-terminal side of aspartic acid (Asp).
- Acids include organic acids and inorganic acids.
- organic acids include formic acid, acetic acid, propionic acid, hexanoic acid, citric acid, chloroacetic acid, dichloroacetic acid, trichloroacetic acid, trifluoroacetic acid (TFA), benzoic acid, salicylic acid, oxalic acid, succinic acid, malonic acid,
- carboxylic acids such as phthalic acid, tartaric acid, malic acid and glycolic acid
- sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and trifluoromethanesulfonic acid.
- inorganic acid examples include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, tetrafluoroboric acid, perchloric acid, periodic acid and the like. These acids may be used alone or in combination of two or more. Among these cleaving substances, any one selected from the group consisting of endopeptidase ASP-N, formic acid, acetic acid, trifluoroacetic acid, propionic acid, and combinations thereof is preferable.
- an acid When an acid is used as a cleaving substance, it is usually preferable to use 10 ⁇ L to 3 mL of 1 to 2% of organic acid, more preferably 100 ⁇ L to 1 mL, per 5 nmol of glucagon / secretin family peptide.
- the acidity upon cleavage is preferably pH 1 to 5, more preferably pH 2 to 4.
- the peptide bond on the C-terminal side or N-terminal side of aspartic acid (Asp) can be hydrolyzed, and analysis and quantification can be performed using the peptide fragment on the N-terminal side of the hydrolyzed glucagon / secretin family peptide.
- the reaction temperature is usually 25 to 45 ° C., preferably 35 to 40 ° C.
- the reaction time is usually 4 to 24 hours, preferably 10 to 18 hours.
- the reaction temperature is usually 60 to 120 ° C., preferably 95 to 110 ° C.
- the reaction time is usually 30 minutes to 24 hours, preferably 2 to 20 hours.
- a solvent can be used in any reaction, and any solvent may be used as long as it does not inhibit the reaction.
- glucagon / secretin family peptides when the peptide bond of aspartic acid (Asp) is cleaved, by measuring the N-terminal peptide fragment, two or more types of glucagon / secretin family peptides are distinguished, and It can be analyzed at the same time. In this case, it is preferable to select two or more glucagon / secretin family peptides having the same extraction conditions.
- the amino acid sequence on the N-terminal side is important for the physiological activity of glucagon / secretin family peptides. That is, some glucagon / secretin family peptides lose activity due to the loss of 2 amino acid residues on the N-terminal side by enzymes such as Dipeptidil peptidase-4 (hereinafter referred to as DPP-4).
- DPP-4 Dipeptidil peptidase-4
- GIP 1-42 which is an active GIP, is cleaved at the N-terminal two residues (position 1 Tyr and position 2 Ala) by DPP-4 to become GIP 3-42 and lose activity.
- the active glucagon / secretin family peptide retaining the N-terminal two residues and the inactive having lost the N-terminal two residues It can be quantified separately from the type glucagon / secretin family peptides. If the active type and the non-active type can be distinguished and quantified, both the active and non-active glucagon / secretin family peptides can be quantified simultaneously.
- the number of amino acids in the peptide to be measured is preferably 5 to 14, more preferably 6 to 9.
- the peptide fragment in this method falls within these numerical ranges and is suitable for measurement by mass spectrometry.
- N-terminal peptide fragment of GIP or an analog thereof can be represented by the following formula.
- X 1 is Tyr, D-Tyr, N-Acetyl-Tyr, N-Pyroglutamyl-Tyr, N-Glucitol-Tyr, N-Palmitate-Tyr, N-Fmoc-Tyr, N-alkyl-Tyr, N-glycosyl- Tyr, N-isopropyl-Tyr or deletion
- X 2 is Ala, D-Ala, Gly, Ser, 2-Aminobutylic acid, Aminoisobutylic acid, Phosphoserine, Sarcosine, or deletion
- X 3 is Glu, D-Glu, Pro, Hydroxyproline, Lys, Tyr, or Phe
- X 4 is Asp
- N-terminal peptide fragment of GIP or its analog examples include the following.
- a non-active peptide fragment is the one lacking the two amino acid residues on the N-terminal side of the N-terminal peptide fragment of the following GIP or its analog.
- Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser [SEQ ID NO: 1], N-Acetyl-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser [N-Acetyl variant of SEQ ID NO: 1] ], N-Pyroglutamyl-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser [N-Pyroglutamyl variant of SEQ ID NO: 1], N-Glucitol-Tyr-Ala-Glu-Gly-Thr-Phe-Ile -Ser [N-Glucitol variant of SEQ ID NO: 1], N-Palmitate-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser [N-Palmitate variant of SEQ ID NO: 1], N-Fmoc-Tyr -Ala-Glu-Gly-Thr-Phe-Ile-Ser [N-Fmoc variant of
- Tyr-Sar-Glu-Gly-Thr-Phe-Ile-Ser-Asp [SEQ ID NO: 96] Tyr-Ala-Pro-Gly-Thr-Phe-Ile-Ser-Asp [SEQ ID NO: 97], Tyr-Ala-Hyp-Gly-Thr-Phe-Ile-Ser-Asp [SEQ ID NO: 98], Tyr-Ala-Lys-Gly-Thr-Phe-Ile-Ser-Asp [SEQ ID NO: 99], Tyr-Ala- Tyr-Gly-Thr-Phe-Ile-Ser-Asp [SEQ ID NO: 100], Tyr-Ala-Phe-Gly-Thr-Phe-Ile-Ser-Asp [SEQ ID NO: 100] 101],
- N-terminal peptide fragment of GLP-1 or an analog thereof can be represented by the following formula.
- N-terminal peptide fragment of GLP-1 or an analog thereof include the following.
- a non-active peptide fragment is the one lacking the two amino acid residues on the N-terminal side of the N-terminal peptide fragment of the following GLP-1 or its analog.
- N-terminal peptide fragment of GLP-2 include the following.
- a non-active peptide fragment is the one lacking the two amino acid residues on the N-terminal side of the N-terminal peptide fragment of GLP-2 shown below. His-Ala-Asp-Gly-Ser-Phe-Ser [SEQ ID NO: 110], His-Ala-Asp-Gly-Ser-Phe-Ser-Asp [SEQ ID NO: 111]
- N-terminal peptide fragment of Glucagon include the following. His-Ser-Gln-Gly-Thr-Phe-Thr-Ser [SEQ ID NO: 11], His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp [SEQ ID NO: 12]
- the peptide fragment of the glucagon / secretin family peptide in the sample is subjected to a treatment for removing contaminants such as proteins, peptides, and low-molecular substances in the body, which are present in the sample, prior to the analysis process by the mass spectrometer. It is preferable. If this processing is performed, the quantitative sensitivity or accuracy in the analysis step can be further increased.
- a person skilled in the art can appropriately perform a treatment for removing these contaminant components by combining one or more of concentration, phase transfer, extraction, crystallization, centrifugation, and purification means.
- the treatment for removing the contaminant components can be performed either before or after the cutting step, or both.
- Examples of the extraction method performed as a treatment for removing the contaminating component include a solid phase extraction method.
- the solid-phase extraction method can be carried out by a known method such as holding (adsorbing) the glucagon / secretin family peptide on the solid phase and then eluting the glucagon / secretin family peptide held (adsorbed) on the solid phase with an elution solvent.
- These solid phases can be used alone or in combination of two or more.
- solid phases having different properties such as a combination of an ion exchange type solid phase extraction tool and a reverse phase type solid phase extraction tool are used. Then, many kinds of contaminating components can be efficiently removed.
- An anion exchange type solid phase extraction tool or a cation exchange type solid phase extraction tool can be used, and those skilled in the art can appropriately select the type depending on the type of glucagon / secretin family peptide.
- a commercially available cartridge or minicolumn filled with the above solid phase or a 96-well type solid phase extraction plate or the like can be used.
- the concentration operation can be performed by, for example, vacuum concentration.
- the purification performed as a treatment for removing the contaminating components can be performed by liquid chromatography or the like.
- liquid chromatography two phases, a mobile phase and a stationary phase, are involved.
- the mobile phase is a water-acetonitrile mixture
- the stationary phase is an octadecylsilyl group (C18) bonded to a silica gel carrier.
- C18 octadecylsilyl group
- the flow rate of liquid chromatography is preferably 50 nL / min to 50 ⁇ L / min, more preferably 100 nL / min to 1 ⁇ L / min. If the flow rate is low, extremely fine charged droplets can be created during electroion spray ionization, and highly efficient desolvation is possible.
- the inner diameter of the column used in liquid chromatography is preferably 50 ⁇ m or more and less than 1 mm, more preferably 50 ⁇ m or more and 800 ⁇ m or less. If a column with a small inner diameter is used, diffusion of peptide fragments to be measured in the column can be prevented, and measurement sensitivity can be improved.
- Peptide fragments separated and eluted by liquid chromatography are measured with a mass spectrometer.
- mass spectrometry a substance to be measured is first ionized by some method, and the mass / charge ratio (m / z) of the ions and the ion amount of the mass are measured. Therefore, there are various combinations of ionization and ion measurement methods in mass spectrometers.
- Mass spectrometers (MS / MS, MS / MS / MS, etc.) combined with two or more mass spectrometers, tandem mass spectrometer (tandem MS) mass A triple quadrupole mass spectrometer (LC / MS / MS, etc.) with an analyzer and two transmission-type quadrupole mass spectrometers in series, with a collision cell between them, and mass spectrometry using the ion trap function
- Mass spectrometers having various functions such as a meter (MS / MS / MS, etc.) or a quadrupole time-of-flight mass spectrometer are known.
- LC / MS / MS selects a precursor ion specific to the peptide to be quantified, then collides with argon or the like to dissociate the ions to generate a new ion group, and one or more from this new ion group (product ion) Multiple ions are analyzed with a mass spectrometer (MS).
- MS mass spectrometer
- MS / MS / MS can measure by setting primary product ions generated from precursor ions and secondary product ions generated from primary product ions, so it is advantageous for measurement in a system with similar amino acid sequences. It is.
- the precursor ion is an ion generated by electrospray ionization, atmospheric pressure chemical ionization or the like, and is a precursor ion of product ions.
- the separation step and the analysis step can be performed by a method comprising a combination of liquid chromatography and mass spectrometry.
- Specific examples of such methods include liquid chromatography / tandem mass spectrometry (LC / MS / MS, LC / MS / MS / MS, etc.), liquid chromatography / mass spectrometry (LC / MS), and the like.
- LC / MS liquid chromatography / tandem mass spectrometry
- LC / MS liquid chromatography / mass spectrometry
- Mass spectrum is a spectrum obtained by mass spectrometry of peptide fragments, with the horizontal axis representing m / z and the vertical axis representing detection intensity.
- m / z is a value obtained by dividing the mass of an ion by the unit of unitary atomic mass and further by the number of electrifications of the ion. Since the mass spectrometer can specifically introduce only m / z ions to the detector by inputting m / z specific to the substance to be measured to the apparatus, analysis with high specificity is possible.
- Table 1 An example of a peptide fragment of the glucagon / secretin family peptide is shown in Table 1.
- m / z of a unique precursor ion can be set by a method well known to those skilled in the art.
- MS / MS or MS / MS / MS is used, 1 to 5 fragment ions, preferably 1 to 3 m / z, can be set in the same manner, and highly specific quantification can be performed.
- a glucagon / secretin family peptide or its analog in a sample is quantified using a calibration curve.
- a calibration curve can be prepared according to a method generally used by those skilled in the art by measuring a peptide fragment derived from a standard substance using a standard solution diluted in stages and measuring it with a mass spectrometer.
- the calibration curve preferably has a trueness within ⁇ 20% at the lower limit concentration, within ⁇ 15% at other concentrations, and a correlation coefficient of 0.990 or more.
- human endogenous glucagon / secretin family peptides contained in the sample can be measured, and can be quantified even in a concentration range below the standard concentration.
- the lower limit of quantification is the lowest concentration of the standard substance in the sample to which the standard substance used for preparing the calibration curve is added, meaning that the accuracy is within ⁇ 20%.
- the accuracy of the calibration curve is calculated by dividing the value obtained by subtracting the addition concentration from the quantitative value by the addition concentration.
- the correlation coefficient can be calculated using spreadsheet software.
- the glucagon / secretin family peptide contained in the sample can be quantified using the prepared calibration curve.
- the internal standard substance for example, one obtained by substituting one or more amino acids of the glucagon / secretin family peptide to be measured with a stable isotope-labeled amino acid is preferable. More preferably, any amino acid selected from positions 1 to 15 of the amino acid to be measured is substituted with a stable isotope-labeled amino acid.
- a peptide labeled with a stable isotope for example, Phe can be labeled with a stable isotope to obtain an internal standard substance. Phe is often present in any of positions 1 to 15 of the glucagon / secretin family peptide.
- Phe exists in the 6th position of GIP 1-42 , the 12th position of GLP-1 (7-36), the 6th position of GLP-2, and the 6th position of Glucagon.
- Phe is an amino acid suitable for labeling with a stable isotope and can have a difference of 10 Da or more from the original peptide to be measured, thus minimizing crosstalk during quantification by mass spectrometry .
- Leu, Ile, Val, Ala, Tyr, Glu, Gly, Thr, Ser or Pro are also suitable amino acids for labeling with a stable isotope.
- stable isotopes include 2 H, 13 C, 15 N and 18 O, and these can be used alone or in combination of two or more.
- a method of introducing 18 O by performing hydrolase cleavage in H 2 18 O there are a method of performing stable isotope labeling using a commercially available stable isotope labeling reagent, and the like.
- the stable isotope-labeled peptide is chemically identical except that the labeled amino acid is different in mass from the labeled amino acid and exhibits the same behavior in LC-MS / MS measurement, thus eliminating the influence on the ionization of contaminant components.
- a mass difference is generated between the measurement target substance and the internal standard substance, it is detected as another peak by LC / MS or the like, and quantification is possible based on the ratio of the area or height of both peaks.
- the internal standard substance labeled with a stable isotope may be the entire amino acid sequence of the glucagon / secretin family peptide or a peptide fragment to be measured.
- an internal standard substance in which a part of the entire amino acid sequence is labeled is preferable. If an internal standard substance labeled with a part of the peptide fragment to be measured is used, the extraction efficiency is not always the same as that of the glucagon / secretin family peptide to be quantified in the pretreatment up to the cleavage step. . In addition, digestion efficiency with a sequence-specific protease or acid is not necessarily the same as that of the glucagon / secretin family peptide to be quantified.
- a peptide labeled with a stable isotope but also a peptide in which one or more amino acids of a peptide fragment to be measured are modified, or a peptide in which the amino acid sequence of the peptide fragment to be measured is changed is changed. It can also be used.
- kits for quantifying a glucagon / secretin family peptide or an analog thereof in a sample includes (a) a control matrix, (b) an internal standard substance or a solution thereof, (c) a glucagon / secretin family peptide or a solution thereof, (d) a cleaving substance, and (e) a solid phase extraction plate.
- Said (d) cleaving substance is as described in paragraph 24, but any one selected from the group consisting of endopeptidase ASP-N, formic acid, acetic acid, trifluoroacetic acid, propionic acid, and combinations thereof is preferred. .
- a buffer for treating the cleaving substance can be added to the kit as necessary.
- the method of using this kit will be described below.
- the internal standard substance or a solution thereof is added to the sample and (a) the control matrix.
- a predetermined amount of glucagon / secretin family peptide or a solution thereof is added.
- a cleaved substance is used to obtain a peptide fragment, and
- an extraction operation is performed using a solid phase extraction plate.
- the glucagon / secretin family peptide in the sample can be quantified.
- GIP 1-8 The amino acid sequence of GIP 1-8 is Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser [SEQ ID NO: 1].
- GIP 1-16 16 of lysine GIP 1-42 was used GIP 1-16 truncated at the C-terminal side. When GIP 1-42 is cleaved with Trypsin, lysine at position 16 is cleaved on the C-terminal side.
- the amino acid sequence of GIP 1-16 is Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys [SEQ ID NO: 112].
- the peak changing with time was a peak of an oxidant obtained by oxidizing methionine (Met) of GIP 1-16 .
- the fact that the peak increases with time indicates that the molecular weight of the peptide changes with time, and the concentration in the sample cannot be measured accurately.
- the peptide fragment of GIP 1-8 was more stable than the peptide fragment of GIP 1-16 .
- GIP analogue is a method glucagon-secretin family peptides (2S-GIP-NH 2) shows an embodiment for measuring the [C-terminal amide derivative of SEQ ID NO: 113].
- This GIP analog is a GIP analog obtained by substituting serine for alanine at the 2-position of active GIP and amidating the C-terminus. ASP-N was used as the cleavage material.
- the GIP analog was dissolved in 50 mM Tris / HCl and 2.5 mM ZnSO 4 solution (pH 8.0). 1 ⁇ g of Asp-N was added to this solution and incubated at 37 ° C. for 15 hours to cleave the GIP analog.
- GIP measurement method The method illustrates one embodiment for measuring the glucagon-secretin family peptides and is active GIP [SEQ ID NO: 13].
- Formic acid was used as the cleavage material.
- Active GIP 1-42 was dissolved in purified water to make a 100 ⁇ M solution. To 50 ⁇ L of this solution, 500 ⁇ L of 2% formic acid was added and incubated at 108 ° C. for up to 18 hours, and the cleavage of active GIP 1-42 was confirmed over time. Thereafter, each sample was analyzed by LC / MS (LCQ Deca XP Plus). Results The activated GIP 1-42 was cleaved with formic acid, and the analysis results are shown in FIG.
- Example 3 GIP measurement method Another embodiment for measuring active GIP [SEQ ID NO: 13] which is a glucagon / secretin family peptide is shown. Acetic acid was used as the cleavage material. The analysis was performed in the same manner as in Example 2 except that 500 ⁇ L of 2% acetic acid was added instead of 500 ⁇ L of 2% formic acid. The analysis results are shown in FIG.
- GIP 1-8 [SEQ ID NO: 1] in which the N-terminal side of aspartic acid at position 9 of active GIP 1-42 was cleaved, and GIP in which the C-terminal side of aspartic acid at position 9 in active GIP 1-42 was cleaved A peak of 1-9 [SEQ ID NO: 2] was observed.
- Example 4 GIP measurement method Another embodiment for measuring active GIP [SEQ ID NO: 13], which is a glucagon / secretin family peptide, is shown. Trifluoroacetic acid was used as the cleavage material. The analysis was performed in the same manner as in Example 2 except that 500 ⁇ L of 1% trifluoroacetic acid was added instead of 500 ⁇ L of 2% formic acid. The analysis results are shown in FIG.
- GIP 1-8 [SEQ ID NO: 1] in which the N-terminal side of aspartic acid at position 9 of active GIP 1-42 was cleaved, and GIP in which the C-terminal side of aspartic acid at position 9 in active GIP 1-42 was cleaved 1-9
- GLP-1 measurement method Method Shows one embodiment for measuring glucagon secretin family peptide GLP-1 (7-36) [SEQ ID NO: 25].
- ASP-N was used as the cleavage material.
- Analysis was performed in the same manner as in Example 1 except that GLP-1 (7-36) was used instead of the GIP analog.
- the result analysis results are shown in FIG.
- a peak of GLP-1 (7-14) in which the N-terminal side of aspartic acid at position 15 of GLP-1 (7-36) was cleaved was observed (m / z 425 ([M + 2H] 2+ )).
- Glucagon measurement method Shows an embodiment for measuring the Glucagon [SEQ ID NO: 69] is a method glucagon-secretin family peptides. ASP-N was used as the cleavage material. Analysis was performed in the same manner as in Example 1 except that Glucagon was used instead of the GIP analog. The result analysis results are shown in FIG. A peak of Glucagon 1-8 in which the N-terminal side of aspartic acid at position 9 of Glucagon was cleaved was observed (m / z 864 ([M + H] + )). Glucagon 1-8 : His-Ser-Gln-Gly-Thr-Phe-Thr-Ser [SEQ ID NO: 11]
- Example 7 Method for measuring GLP-1 analog Shows an embodiment for measuring GLP-1 analogue is the method glucagon-secretin family peptides (8S-GLP-1) [SEQ ID NO: 31]. This analog is a GLP-1 analog in which the alanine at position 8 of GLP-1 (7-36) is replaced with serine. ASP-N was used as the cleavage material. The analysis was conducted in the same manner as in Example 1, except that 5 nmol of GIP analog was replaced with 5 nmol of GLP-1 and the amount of Asp-N was changed from 1 ⁇ g to 2 ⁇ g. The result analysis results are shown in FIG.
- 8S-GLP-1 (7-14) His-Ser-Glu-Gly-Thr-Phe-Thr-Ser [SEQ ID NO: 10]
- the glucagon / secretin family can be used regardless of the type of glucagon / secretin family peptide by cleaving the peptide bond of any aspartic acid selected from positions 1 to 14 of the glucagon / secretin family peptide.
- the peptide fragment of the peptide could be analyzed well.
- Example 8 Method for quantifying active GIP An embodiment in which the concentration of active GIP [SEQ ID NO: 13] in a sample is quantified using a calibration curve is shown.
- Method 1 Preparation of calibration curve samples The following internal standards were synthesized as internal standard substances in which Phe at position 6 of GIP was replaced with 13 C 9 , 15 N-Phe, which is a stable isotope-labeled amino acid. A sample for a calibration curve was prepared according to the following procedures (1) to (3).
- This solution was diluted to prepare 20.0 pM, 200 pM, 2.00 nM, 20.0 nM, and 1.00 ⁇ M standard solutions.
- (2) Preparation of internal standard substance standard solution The synthesized internal standard substance was dissolved to prepare a 100 ⁇ M solution. This solution was diluted to prepare 2.00 nM, 20.0 nM, and 1.00 ⁇ M internal standard substance standard solutions.
- Example 9 Simultaneous reproducibility test of active GIP in human plasma Method
- human plasma active GIP [SEQ ID NO: 13] was quantified, and the concentration of each sample was calculated by applying the obtained peak area ratio to each calibration curve.
- the accuracy (Bias%) and accuracy (% CV) of the obtained measured value (n 3) were calculated.
- Judgment criteria were accuracy within ⁇ 15% (quantitative limit within ⁇ 20%) and accuracy within 15% (quantitative limit within 20%). The results are shown in Table 5.
- the active GIP at each concentration met the criteria for simultaneous reproducibility. This indicates that the reproducibility of the quantitative value is good.
- Example 10 Method for simultaneous determination of active GIP and non-active GIP Method 1.
- sample for calibration curve In the same manner as in Example 8, a measurement sample containing a standard substance and an internal standard substance was prepared.
- an internal standard substance As an internal standard substance, the following peptide was synthesized and added by replacing Phe at position 6 of GIP with 13 C 9 , 15 N-Phe.
- the inactive GIP internal standard substance is one in which the N-terminal 2 residues of the active GIP internal standard substance are missing.
- This solution was diluted with 50% ethanol to prepare 20.0 pM, 200 pM, 2.00 nM, 20.0 nM, and 1.00 ⁇ M standard solutions.
- (2) Preparation of internal standard substance standard solution for active GIP The synthesized internal standard peptide was dissolved to prepare a 100 ⁇ M solution. This solution was diluted to prepare 1.00 nM, 10.0 nM, and 1.00 ⁇ M standard solutions.
- (3) Preparation of non-active GIP standard solution The purchased non-active GIP was dissolved to prepare a 100 ⁇ M solution. This solution was diluted to prepare 20.0 pM, 200 pM, 2.00 nM, 20.0 nM, and 1.00 ⁇ M standard solutions.
- a peptide fragment derived from an inactive GIP (Glu-Gly-Thr-Phe-Ile-Ser [SEQ ID NO: 3]) and a peptide fragment derived from an internal standard for inactive GIP (Glu-Gly-Thr- 13C ) 9 , 15 N-Phe- Ile-Ser (amino acid sequence is the same as SEQ ID NO: 3)) could be detected as independent peaks (FIG. 13).
- the linearity regression of the active GIP concentration and the peak area ratio (peak area of active GIP-derived peptide / peak area of internal standard substance-derived peptide) with the linear least squares method gives the slope, intercept, and correlation coefficient (r). Asked.
- the correlation coefficient (r) was 0.990 or more when the weighting 1 / x was set (FIG. 14).
- the linearity regression of the non-active GIP concentration and the peak area ratio between them (peak area of non-active GIP-derived peptide / peak area of internal standard substance-derived peptide for non-active GIP) by linear least squares method, slope, intercept and Correlation coefficient (r) was determined.
- the weighting 1 / x was set and the correlation coefficient (r) was 0.990 or more (FIG. 15).
- Example 11 Simultaneous determination of active GIP non-active GIP in diabetic patients [alpha] -glucosidase inhibitor (50 mg, 3 times a day) Active GIP in plasma before and 1 hour after meal in 6 diabetic patients [sequence] No. 13] and inactive GIP [SEQ ID NO: 14] were simultaneously quantified.
- Sample Preparation (1) Preparation of Sample for Calibration Curve Samples were prepared as shown in Table 9 in the same manner as in Example 9.
- results A plasma sample of a diabetic patient was measured, and the peptide fragment derived from the active GIP [SEQ ID NO: 1] and the peptide fragment derived from the internal standard for active GIP (amino acid sequence is the same as SEQ ID NO: 1) as independent peaks It was detected (FIG. 16). Similarly, inactive GIP could be detected as independent peaks (FIG. 17). Weighting 1 / x in the range of 1pM to 500pM by linear regression of the active GIP concentration and the peak area ratio between them (peak area of active GIP-derived peptide / peak area of internal standard substance-derived peptide) using the linear least squares method A calibration curve was created by setting.
- the peak area ratio (peak area of active GIP-derived peptide / peak area of internal standard substance-derived peptide) obtained from a diabetic patient plasma sample was fitted to this calibration curve, and the plasma active GIP concentration was calculated.
- a weighting 1 / x was set in the range of 10 pM to 500 pM, a calibration curve was created in the same manner as the active GIP, and the concentration of non-active GIP in the plasma of diabetic patients was calculated.
- the results are shown in Table 13. The numerical values in the table indicate the mean plasma concentrations ⁇ standard deviation of each.
- a calibration curve can be prepared in the same manner as in Example 8 to Example 11, and the glucagon / secretin family peptide in the sample can be quantified using the calibration curve. . That is, a peptide to be quantified is extracted together with an internal standard substance in which a part of the amino acid is substituted with a stable isotope label, and 1 to 7 glucagon / secretin family peptides are extracted in the extraction process as shown in Examples 1 to 7. A peptide fragment is obtained by cleaving the peptide bond of any aspartic acid selected from position 14.
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Abstract
Description
(1)試料中に含まれるグルカゴン・セクレチンファミリーペプチドのアミノ酸配列中に存在するアスパラギン酸のペプチド結合を切断し、グルカゴン・セクレチンファミリーペプチドをペプチド断片にする切断工程;前記切断工程で切断されたペプチド断片を液体クロマトグラフィーにより分離精製し、測定対象物質であるグルカゴン・セクレチンファミリーペプチドのN末端側のペプチド断片を選定する分離精製工程;前記分離精製されたペプチド断片を質量分析し、グルカゴン・セクレチンファミリーペプチドのN末端側のペプチド断片を検出する分析工程;を含む、試料中に含まれるグルカゴン・セクレチンファミリーペプチドの分析方法。
(2)前記切断工程において、配列特異的切断プロテアーゼ又は酸から選択される切断物質を用いてアスパラギン酸のペプチド結合を切断する、(1)に記載の分析方法。
(3)前記切断物質が、エンドペプチダーゼASP-N、ギ酸、酢酸、トリフルオロ酢酸、プロピオン酸、及びそれらの組み合わせからなる群から選択される、(2)に記載の分析方法。
(4)前記分離精製工程において、液体クロマトグラフィーが、流速50nL/min~50μL/minの液体クロマトグラフィーである、(1)~(3)のいずれかに記載の分析方法。
(5)前記グルカゴン・セクレチンファミリーペプチドが、グルコース依存性インスリン分泌刺激ポリペプチド、グルカゴン様ペプチド-1、グルカゴン様ペプチド-2、グルカゴン、及びそれらの類縁体からなる群から選択される、(1)~(4)のいずれかに記載の分析方法。
(6)前記分離精製工程において、プレカーサーイオンの質量選択により測定対象物質を選定する、(1)~(5)のいずれかに記載の分析方法。
(7)さらに、前記分離精製工程において、前記ペプチド断片を固相抽出する工程を含む、(1)~(6)のいずれかに記載の分析方法。
(8)前記分離精製工程及び分析工程が、高速液体クロマトグラフィー/マススペクトロメトリー/マススペクトロメトリー/マススペクトロメトリーにより行われる、(1)~(7)のいずれかに記載の分析方法。
(9)(1)~(8)のいずれかに記載の分析方法を用いて検量線を作成し、前記検量線を用いて、試料中のグルカゴン・セクレチンファミリーペプチドを定量する、試料中のグルカゴン・セクレチンファミリーペプチドの定量方法。
(10)2種以上のグルカゴン・セクレチンファミリーペプチドのペプチド断片を、同時にそれぞれ測定することにより、試料中の2種以上のグルカゴン・セクレチンファミリーペプチドを区別して同時に定量する、(9)に記載の定量方法。
(11)活性型グルカゴン・セクレチンファミリーペプチドのペプチド断片と、非活性型グルカゴン・セクレチンファミリーペプチドのペプチド断片とを、同時にそれぞれ測定することにより、試料中のグルカゴン・セクレチンファミリーペプチドの活性型と非活性型とを区別して同時に定量する、(9)又は(10)に記載の定量方法。
(12)前記検量線を、安定同位体で標識された内標準物質を試料に加えて作成する、(9)~(11)のいずれかに記載の定量方法。
(13)測定対象のペプチド断片及び内標準物質のペプチド断片の各ピーク面積比を用いて定量する、(12)に記載の定量方法。
(14)前記内標準物質が、グルカゴン・セクレチンファミリーペプチドのアミノ酸配列において1位~15位から選択される1又はそれ以上のアミノ酸を、安定同位体で標識されたアミノ酸で置換したペプチドである、(12)又は(13)に記載の定量方法。
(15)(a)コントロール用マトリクス、(b)内標準物質又はその溶液、(c)グルカゴン・セクレチンファミリーペプチド又はそれらの標準物質、若しくはそれらの溶液、(d)切断物質、(e)固相抽出プレートを含む、グルカゴン・セクレチンファミリーペプチドの定量用キット。
(16)前記切断物質が、エンドペプチダーゼASP-N、ギ酸、酢酸、トリフルオロ酢酸、プロピオン酸、及びそれらの組み合わせからなる群から選択される、(15)に記載の定量方法。
(17)前記グルカゴン・セクレチンファミリーペプチドが、グルコース依存性インスリン分泌刺激ポリペプチド、グルカゴン様ペプチド-1、グルカゴン様ペプチド-2、グルカゴン、及びそれらの類縁体からなる群から選択される、(15)又は(16)に記載の定量方法。
Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile-His-Gln-Gln-Asp-Phe-Val-Asn-Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-Ile-Thr-Gln[配列番号13]
[N-Acetyl]-GIP[配列番号13のN-Acetyl変異体]、 [N-Pyroglutamyl]-GIP[配列番号13のN-Pyroglutamyl変異体]、 [N-Glucitol]-GIP[配列番号13のN-Glucitol変異体]、 [N-Palmitate]-GIP[配列番号13のN-Palmitate変異体]、 [N-Fmoc]-GIP[配列番号13のN-Fmoc変異体]、 [N-alkyl]-GIP[配列番号13のN-alkyl変異体]、 [N-glycosyl]-GIP[配列番号13のN-glycosyl変異体]、 [N-isopropyl]-GIP[配列番号13のN-isopropyl変異体]、 [Gly2]-GIP[配列番号15]、 [D-Ala2]-GIP[配列番号16](但し、D-Alaは、D型Alanineを意味する。下記においても同じ)。、 [Aib2]-GIP[配列番号17](但し、Aibは、Aminoisobutylic acidを意味する。下記においても同じ。)、 [Phosphoserine2]-GIP[配列番号18]、 [Sar2]-GIP[配列番号19](但し、Sarは、N-Methylglycine(MeGly), sarcosineを意味する。下記においても同じ。)、 [Pro3]-GIP[配列番号20]、 [Hyp3]-GIP[配列番号21](但し、Hypは、Hydroxyprolineを意味する。下記においても同じ。)、 [Lys3]-GIP[配列番号22]、 [Tyr3]-GIP[配列番号23]、 [Phe3]-GIP[配列番号24]、[Ser2]-GIP[配列番号113]
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly[配列番号26]
[Ser8]-GLP-1(7-37)[配列番号31]、[Gly8]-GLP-1(7-37) [配列番号32]、[Val8]-GLP-1(7-37) [配列番号33]、[Glu22]-GLP-1(7-37) [配列番号34]、[Lys22]-GLP-1(7-37) [配列番号35]、[Val8,Glu22]-GLP-1(7-37) [配列番号36]、[Val8,Lys22]-GLP-1(7-37) [配列番号37]、[Gly8,Glu22]-GLP-1(7-37) [配列番号38]、[Gly8,Lys22]-GLP-1(7-37) [配列番号39]、[Val8,Glu30]-GLP-1(7-37) [配列番号40]、[Gly8,Glu30]-GLP-1(7-37) [配列番号41]、[Val8,His37]-GLP-1(7-37) [配列番号42]、[Gly8,His37]-GLP-1(7-37) [配列番号43]、[Arg34]-GLP-1(7-37) [配列番号44]、[Lys18]-GLP-1(7-37) [配列番号45]、[Gly8,Glu22,Gly36]-GLP-1(7-37) [配列番号46]、[Aib8,Aib22]-GLP-1(7-37) [配列番号47]、[Aib8,Aib35]-GLP-1(7-37) [配列番号48]、[Aib8,Aib22,Aib35]-GLP-1(7-37) [配列番号49]、[Glu22,Glu23]-GLP-1(7-37) [配列番号50]、[Gly8,Glu22,Glu23]-GLP-1(7-37) [配列番号51]、[Val8,Glu22,Glu23]-GLP-1(7-37) [配列番号52]、[Val8,Glu22,Val25]-GLP-1(7-37) [配列番号53]、[Val8,Glu22,Ile33]-GLP-1(7-37) [配列番号54]、[Val8,Glu22,Val25,Ile33]-GLP-1(7-37) [配列番号55]、及びこれらの37位が欠失したGLP-1(7-36)型、並びにこれらの36位と37位とが欠失したGLP-1(7-35)型
[Ser2]-GLP-2[配列番号66]、[Gly2]-GLP-2 [配列番号67]、[Val2]-GLP-2[配列番号68]
[Arg12]-Glucagon[配列番号70]、[Arg12,Lys20]-Glucagon[配列番号71]、[Arg12,Lys24]-Glucagon[配列番号72]、[Arg12,Lys29]-Glucagon[配列番号73]、[Glu9]-Glucagon[配列番号74]、[Glu9,Glu16,Lys29]-Glucagon[配列番号75]、[Lys13,Glu17]-Glucagon[配列番号76]、[Glu20,Lys24]-Glucagon[配列番号77]
式:X1-X2-X3-Gly-Thr-Phe-Ile-Ser-X4[配列番号78]
[式中、
X1は、Tyr、D-Tyr、N-Acetyl-Tyr、N-Pyroglutamyl-Tyr、N-Glucitol-Tyr、N-Palmitate-Tyr、N-Fmoc-Tyr、N-alkyl-Tyr、N-glycosyl-Tyr、N-isopropyl-Tyr又は欠失、
X2は、Ala、D-Ala、Gly、Ser、2-Aminobutylic acid、Aminoisobutylic acid、Phosphoserine、Sarcosine、又は欠失、
X3は、Glu、D-Glu、Pro、Hydroxyproline、Lys、Tyr、又はPhe、
X4は、Asp又は欠失、
を意味する。]
Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser[配列番号1]、N-Acetyl-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser[配列番号1のN-Acetyl変異体]、N-Pyroglutamyl-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser[配列番号1のN-Pyroglutamyl変異体]、N-Glucitol-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser[配列番号1のN-Glucitol変異体]、N-Palmitate-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser[配列番号1のN-Palmitate変異体]、N-Fmoc-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser[配列番号1のN-Fmoc変異体]、N-alkyl-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser[配列番号1のN-alkyl変異体]、N-glycosyl-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser[配列番号1のN-glycosyl変異体]、N-isopropyl-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser[配列番号1のN-isopropyl変異体]、Tyr-Gly-Glu-Gly-Thr-Phe-Ile-Ser[配列番号79]、Tyr-Ser-Glu-Gly-Thr-Phe-Ile-Ser[配列番号5]、Tyr-D-Ala-Glu-Gly-Thr-Phe-Ile-Ser[配列番号80]、Tyr-Abu-Glu-Gly-Thr-Phe-Ile-Ser[配列番号81](但し、Abuは、2-Aminobutylic acidを意味する。下記においても同じ。)、Tyr-Aib-Glu-Gly-Thr-Phe-Ile-Ser[配列番号82]、Tyr-Phosphoserine-Glu-Gly-Thr-Phe-Ile-Ser[配列番号83]、Tyr-Sar-Glu-Gly-Thr-Phe-Ile-Ser[配列番号84]、Tyr-Ala-Pro-Gly-Thr-Phe-Ile-Ser[配列番号85]、Tyr-Ala-Hyp-Gly-Thr-Phe-Ile-Ser[配列番号86]、Tyr-Ala-Lys-Gly-Thr-Phe-Ile-Ser[配列番号87]、Tyr-Ala-Tyr-Gly-Thr-Phe-Ile-Ser[配列番号88]、Tyr-Ala-Phe-Gly-Thr-Phe-Ile-Ser[配列番号89]、Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp[配列番号2]、N-Acetyl-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp[配列番号2のN-Acetyl変異体]、N-Pyroglutamyl-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp[配列番号2のN-Pyroglutamyl変異体]、N-Glucitol-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp[配列番号2のN-Glucitol変異体]、N-Palmitate-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp[配列番号2のN-Palmitate変異体]、N-Fmoc-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp[配列番号2のN-Fmoc変異体]、N-alkyl-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp[配列番号2のN-alkyl変異体]、N-glycosyl-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp[配列番号2のN-glycosyl変異体]、N-isopropyl-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp[配列番号2のN-isopropyl変異体]、Glu-Gly-Thr-Phe-Ile-Ser-Asp[配列番号4]、Tyr-Gly-Glu-Gly-Thr-Phe-Ile-Ser-Asp[配列番号90]、Tyr-Ser-Glu-Gly-Thr-Phe-Ile-Ser-Asp[配列番号91]、Tyr-D-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp[配列番号92]、Tyr-Abu-Glu-Gly-Thr-Phe-Ile-Ser-Asp[配列番号93]、Tyr-Aib-Glu-Gly-Thr-Phe-Ile-Ser-Asp[配列番号94]、Tyr-Phosphoserine-Glu-Gly-Thr-Phe-Ile-Ser-Asp[配列番号95]、Tyr-Sar-Glu-Gly-Thr-Phe-Ile-Ser-Asp[配列番号96]、Tyr-Ala-Pro-Gly-Thr-Phe-Ile-Ser-Asp[配列番号97]、Tyr-Ala-Hyp-Gly-Thr-Phe-Ile-Ser-Asp[配列番号98]、Tyr-Ala-Lys-Gly-Thr-Phe-Ile-Ser-Asp[配列番号99]、Tyr-Ala-Tyr-Gly-Thr-Phe-Ile-Ser-Asp[配列番号100]、Tyr-Ala-Phe-Gly-Thr-Phe-Ile-Ser-Asp[配列番号101]、
式:His-X8-X9-Gly-Thr-Phe-Thr-Ser-X15[配列番号102]
[式中、
X8は、Ala、Gly、Ser、Thr、Leu、Ile、Val、Glu、Lys、又はAib、
X9は、Glu、Gly、又はLys、
X15は、Asp又は欠失、
を意味する。]
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser[配列番号6]、His-Ser-Glu-Gly-Thr-Phe-Thr-Ser[配列番号10]、His-Gly-Glu-Gly-Thr-Phe-Thr-Ser[配列番号103]、His-Val-Glu-Gly-Thr-Phe-Thr-Ser[配列番号104]、His-Aib-Glu-Gly-Thr-Phe-Thr-Ser[配列番号105]、His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp[配列番号7]、His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp[配列番号106]、His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp[配列番号107]、His-Val-Glu-Gly-Thr-Phe-Thr-Ser-Asp[配列番号108]、His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp[配列番号109]
His-Ala-Asp-Gly-Ser-Phe-Ser[配列番号110]、His-Ala-Asp-Gly-Ser-Phe-Ser-Asp[配列番号111]
His-Ser-Gln-Gly-Thr-Phe-Thr-Ser[配列番号11]、His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp[配列番号12]
方法
グルカゴン・セクレチンファミリーペプチドであるGIPの測定対象となるペプチド断片の安定性を比較した。固相合成により合成した下記(a)(b)のペプチド断片に、0.1%TFA-20%アセトニトリルを加えて2.0μM溶液とした。この溶液をLC/MS/MS(Applied Biosystems:QSTAR(登録商標)Elite)にて測定した。測定は、調製直後及び調製後17日目に行った。
(a)GIP1-8:GIP1-42の9位のアスパラギン酸がN末端側で切断されたGIP1-8を用いた。GIP1-8のアミノ酸配列は、Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser[配列番号1]である。
(b)GIP1-16:GIP1-42の16位のリジンがC末端側で切断されたGIP1-16を用いた。尚、GIP1-42をTrypsinで切断すると16位のリジンがC末端側で切断される。GIP1-16のアミノ酸配列は、Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys[配列番号112]である。
調製直後の測定値を図1に示し、調製後17日目の測定値を図2に示す。
GIP1-8では、1つのピークが観測された(m/z887.4([M+H]+))。GIP1-16では、2つのピークが観測された(m/z905.9([M+2H]2+)及びm/z913.9([M+2H]2+))。GIP1-16で観測されたピークの1つは、冷蔵保存した際に経時的に大きくなり、調製後17日目では、もう一方のピークとのピーク強度比はほぼ1:1となった。経時的に変化するピークは、GIP1-16のメチオニン(Met)が酸化された酸化体のピークであった。ピークが経時的に大きくなることは、経時的にペプチドの分子量が変化し、試料中の濃度を正確に測定できないことを示している。GIP1-8のペプチド断片は、GIP1-16のペプチド断片より安定性が勝っていた。
方法
グルカゴン・セクレチンファミリーペプチドであるGIP類縁体(2S-GIP-NH2)[配列番号113のC末端アミド誘導体]を測定する一実施形態を示す。このGIP類縁体は、活性型GIPの2位のアラニンをセリンに置換し、C末端をアミド化したGIP類縁体である。切断物質はASP-Nを用いた。当該GIP類縁体を、50mMのTris/HCl及び2.5mMのZnSO4溶液(pH8.0)で溶解した。この溶液に、Asp-Nを1μg加え、37℃で15時間インキュベートして、当該GIP類縁体を切断した。その後、試料をLC/MS(LCQ Deca XP Plus)で分析した。
結果
GIP類縁体をAsp-Nにより切断し、分析した結果を図3に示す。1つのピーク(m/z 903([M+H]+))が観察された。このピークは、活性型GIP類縁体の9位のアスパラギン酸のN末側が切断された2S-GIP1-8であった。
2S-GIP1-8:Tyr-Ser-Glu-Gly-Thr-Phe-Ile-Ser[配列番号5]
方法
グルカゴン・セクレチンファミリーペプチドである活性型GIP[配列番号13]を測定する一実施形態を示す。切断物質はギ酸を用いた。活性型GIP1-42を精製水で溶解し100μM溶液とした。この溶液50μLに、2%ギ酸を500μL加え、108℃で18時間までインキュベートして、経時的に活性型GIP1-42の切断を確認した。その後、各試料をLC/MS(LCQ Deca XP Plus)で分析した。
結果
活性型GIP1-42をギ酸により切断し、分析した結果を図4に示す。m/z 887([M+H]+)、及びm/z 1002([M+H]+)のピークが観察された。これら2つのピークは、活性型GIP1-42の9位のアスパラギン酸のN末端側が切断されたGIP1-8、及び活性型GIP1-42の9位のアスパラギン酸のC末端側が切断されたGIP1-9であった。
GIP1-8:Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser[配列番号1]
GIP1-9:Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp[配列番号2]
グルカゴン・セクレチンファミリーペプチドである活性型GIP[配列番号13]を測定する他の実施形態を示す。切断物質は酢酸を用いた。2%ギ酸500μLに代えて2%酢酸500μLを加えた他は、実施例2と同様の方法で分析した。分析結果を図5に示す。活性型GIP1-42の9位のアスパラギン酸のN末端側が切断されたGIP1-8[配列番号1]、及び活性型GIP1-42の9位のアスパラギン酸のC末端側が切断されたGIP1-9[配列番号2]のピークが観察された。
グルカゴン・セクレチンファミリーペプチドである活性型GIP[配列番号13]を測定する他の実施形態を示す。切断物質はトリフルオロ酢酸を用いた。2%ギ酸500μLに代えて1%トリフルオロ酢酸500μLを加えた他は、実施例2と同様の方法で分析した。分析結果を図6に示す。活性型GIP1-42の9位のアスパラギン酸のN末端側が切断されたGIP1-8[配列番号1]、及び活性型GIP1-42の9位のアスパラギン酸のC末端側が切断されたGIP1-9[配列番号2]のピークが観察された。
方法
グルカゴン・セクレチンファミリーペプチドであるGLP-1(7-36)[配列番号25]を測定する一実施形態を示す。切断物質はASP-Nを用いた。GIP類縁体に代えてGLP-1(7-36)とした他は、実施例1と同様の方法で分析した。
結果
分析結果を図7に示す。GLP-1(7-36)の15位のアスパラギン酸のN末端側が切断されたGLP-1(7-14)のピークが観察された(m/z 425([M+2H]2+))。
GLP-1(7-14):His-Ala-Glu-Gly-Thr-Phe-Thr-Ser[配列番号6]
方法
グルカゴン・セクレチンファミリーペプチドであるGlucagon[配列番号69]を測定する一実施形態を示す。切断物質はASP-Nを用いた。GIP類縁体に代えてGlucagonとした他は、実施例1と同様の方法で分析した。
結果
分析結果を図8に示す。Glucagonの9位のアスパラギン酸のN末端側が切断されたGlucagon1-8のピークが観察された(m/z 864([M+H]+))。
Glucagon1-8:His-Ser-Gln-Gly-Thr-Phe-Thr-Ser[配列番号11]
方法
グルカゴン・セクレチンファミリーペプチドであるGLP-1類縁体(8S-GLP-1)[配列番号31]を測定する一実施形態を示す。この類縁体は、GLP-1(7-36)の8位のアラニンをセリンに置換したGLP-1類縁体である。切断物質はASP-Nを用いた。GIP類縁体5nmolに代えてGLP-1類縁体5nmolとし、Asp-Nの量を1μgから2μgに代えた他は、実施例1と同様の方法で分析した。
結果
分析結果を図9に示す。GLP-1類縁体の14位のアスパラギン酸のN末端側が切断された8S-GLP-1(7-14)のピークが観察された(m/z 865([M+H]+))。
8S-GLP-1(7-14):His-Ser-Glu-Gly-Thr-Phe-Thr-Ser[配列番号10]
試料中の活性型GIP[配列番号13]の濃度を、検量線を用いて定量する一実施形態を示す。
方法
1.検量線用試料の調製
内標準物質として、GIPの6位のPheが安定同位体ラベル化アミノ酸である13C9,15N-Pheに置換された、以下の内標準物質を合成した。以下(1)~(3)の手順に従い、検量線用の試料を調製した。
[標準物質]
Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile-His-Gln-Gln-Asp-Phe-Val-Asn-Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-Ile-Thr-Gln[配列番号13]
[内標準物質]
Tyr-Ala-Glu-Gly-Thr- 13 C 9 , 15 N-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile-His-Gln-Gln-Asp-Phe-Val-Asn-Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-Ile-Thr-Gln(アミノ酸配列は、配列番号13に同じ)
(1)活性型GIP標準溶液の調製:活性型GIP1-42(ペプチド研究所より購入)を精製水で溶解し、100μM溶液を調製した。この溶液を希釈し、20.0pM、200pM、2.00nM、20.0nM、及び1.00μM標準溶液を調製した。
(2)内標準物質標準溶液の調製:合成した上記内標準物質を溶解し、100μM溶液を調製した。この溶液を希釈し、2.00nM、20.0nM、及び1.00μM内標準物質標準溶液を調製した。
(3)検量線用試料の調製:2mLチューブに、DPP-4阻害剤(Diprotin A、0.3M)2μLを加え、氷冷下で活性炭処理したヒトのEDTA添加血漿200μL、活性型GIP標準溶液、及び内標準物質標準溶液を加え検量線用試料を調製した。活性型GIP標準溶液、及び内標準物質標準溶液の添加割合を表2に示す。尚、DPP-4阻害剤は、前処理過程におけるGIP1-42のGIP3-42への分解、あるいはGIP1-8のGIP3-8への分解を抑制するために添加した。
(1)除タンパク
上記検量線用試料に180mM炭酸アンモニウム溶液100μL及びエタノール900μLを加え、20分氷冷した。遠心分離後上清を濃縮乾固した。
(2)試料中のグルカゴン・セクレチンファミリーペプチドの切断
前記除タンパク後の検量線用試料をプロテアーゼ(ASP-N)によって切断した。前記除タンパク後の検量線用試料に、50mMのTris/HCl、2.5mMのZnSO4溶液(pH8.0)を100μL加え、更に0.1mg/mLのAsp-N水溶液を8μL加え、37℃で16時間インキュベートした。
(3)固相抽出
固相プレート(Waters製;Oasis MAX 96-well μElution Plate)を使用し、上記(2)で得られた検量線用試料に5%アンモニア水300μLを添加し、これをコンディショニングされた固相プレートに負荷した。固相プレートを洗浄後、0.1%ギ酸-75%アセトニトリル50μLで溶出した。溶出液は、HPLC用バイヤルに移し、遠心エバポレータを用いて濃縮乾固した。さらに、0.1%TFA-10%アセトニトリル20μLで再溶解した。
(4)フィルターろ過
再溶解後の試料を遠心式フィルターユニット(0.2μm)で遠心ろ過し、HPLC用試料とした。
前記前処理を施したHPLC用試料5μLをnanoFlowLC-MS/MS/MSに注入し、HPLC用試料に含まれる標準物質及び内標準物質由来ペプチドを測定した。測定条件を表3及び表4に示す。
(1)HPLC条件
検量線用試料を測定し、活性型GIP由来のペプチド断片(Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser[配列番号1])及び内標準物質由来のペプチド断片(Tyr-Ala-Glu-Gly-Thr- 13 C 9 , 15 N-Phe-Ile-Ser(アミノ酸配列は、配列番号1に同じ))をそれぞれ独立したピークとして検出できた(図10)。活性型GIP濃度と両者のピーク面積比(活性型GIP由来ペプチドのピーク面積/内標準物質由来ペプチドのピーク面積)を線形最小二乗法により直線回帰して、傾きと切片及び相関係数(r)を求めた。1pM~500pMの範囲で検量線を作成した結果、相関係数(r)は0.990以上であり、定量限界は1pMであった(図11)。
方法
実施例8と同様にしてヒト血漿中活性型GIP[配列番号13]を定量し、各検量線に、得られたピーク面積比をあてはめて、各サンプルの濃度を算出した。濃度毎に、得られた測定値(n=3)の真度(Bias%)、精度(%CV)を算出した。判定基準は、真度±15%以内(定量限界は±20%以内)、精度15%以内(定量限界は20%以内)であること、とした。
結果
結果を表5に示す。各濃度における活性型GIPは、同時再現性の判定基準を満たした。このことは、定量値の再現性が良好であることを示している。
方法
1.検量線用の試料の調製
実施例8と同様にして、標準物質及び内標準物質を含む測定用試料を調製した。内標準物質として、GIPの6位のPheを13C9,15N-Pheに置換した、以下のペプチドを合成し加えた。尚、非活性型GIPの内標準物質は、活性型GIP内標準物質のN末端2残基が欠除したものである。
[活性型GIPの標準物質]
Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile-His-Gln-Gln-Asp-Phe-Val-Asn-Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-Ile-Thr-Gln[配列番号13]
[非活性型GIP標準物質]
Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile-His-Gln-Gln-Asp-Phe-Val-Asn-Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-Ile-Thr-Gln(GIP3-42:[配列番号14])
[活性型GIPの内標準物質]
Tyr-Ala-Glu-Gly-Thr- 13 C 9 , 15 N-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile-His-Gln-Gln-Asp-Phe-Val-Asn-Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-Ile-Thr-Gln(アミノ酸配列は、配列番号13に同じ)
[非活性型GIPの内標準物質]
Glu-Gly-Thr- 13 C 9 , 15 N-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile-His-Gln-Gln-Asp-Phe-Val-Asn-Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-Ile-Thr-Gln(アミノ酸配列は、配列番号14に同じ)
(1)活性型GIP標準溶液の調製:活性型GIP(ペプチド研究所より購入)に精製水を加えて溶解し、100μM溶液とした。この溶液を50%エタノールで希釈し、20.0pM、200pM、2.00nM、20.0nM、及び1.00μM標準溶液を調製した。
(2)活性型GIP用内標準物質標準溶液の調製:合成した内標準ペプチドを溶解し、100μM溶液を調製した。この溶液を希釈し、1.00nM、10.0nM、及び1.00μM標準溶液を調製した。
(3)非活性型GIP標準溶液の調製:購入した非活性型GIPを溶解し、100μM溶液を調製した。この溶液を希釈し、20.0pM、200pM、2.00nM、20.0nM、及び1.00μM標準溶液を調製した。
(4)非活性型GIP用内標準物質標準溶液の調製:合成した内標準ペプチドを50%エタノールで溶解し、100μM溶液を調製した。この溶液を50%エタノールで希釈し、1.00nM、10.0nM、及び1.00μM標準溶液を調製した。
(5)検量線用試料の調製:2mLチューブにDPP-4阻害剤(Diprotin A、0.3M)2μLを加え、氷冷下で活性炭処理したヒトのEDTA添加血漿200μL、活性型GIP標準溶液、及び活性型GIP用内標準物質標準溶液、ならびに非活性型GIP標準溶液、及び非活性型GIP用内標準物質標準溶液を表6のように加え、検量線用試料を調製した。
実施例8と同様の方法で前処理を行った。
3.測定
実施例8と同様にして測定した。測定条件は表7及び表8に示す。
(1)HPLC条件
検量線用試料を測定し、活性型GIP由来のペプチド断片(Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser[配列番号1])と活性型GIP用内標準物質由来のペプチド断片(Tyr-Ala-Glu-Gly-Thr- 13 C 9 , 15 N-Phe-Ile-Ser(アミノ酸配列は、配列番号1に同じ))をそれぞれ独立したピークとして検出できた(図12)。また、非活性型GIP由来のペプチド断片(Glu-Gly-Thr-Phe-Ile-Ser[配列番号3])と非活性型GIP用内標準物質由来のペプチド断片(Glu-Gly-Thr- 13 C 9 , 15 N-Phe-Ile-Ser(アミノ酸配列は、配列番号3に同じ))をそれぞれ独立したピークとして検出できた(図13)。活性型GIP濃度と両者のピーク面積比(活性型GIP由来ペプチドのピーク面積/内標準物質由来ペプチドのピーク面積)を線形最小二乗法により直線回帰して傾きと切片及び相関係数(r)を求めた。1pM~500pMの範囲で検量線を作成した結果、重み付け1/xを設定して相関係数(r)は0.990以上であった(図14)。非活性型GIP濃度と両者のピーク面積比(非活性型GIP由来ペプチドのピーク面積/非活性型GIP用内標準物質由来ペプチドのピーク面積)を線形最小二乗法により直線回帰して傾きと切片及び相関係数(r)を求めた。10pM~500pMの範囲で検量線を作成した結果、重み付け1/xを設定して相関係数(r)は0.990以上であった(図15)。
α-グルコシダーゼ阻害剤服用(50mg,1日3回)糖尿病患者6例の食前及び食後1時間における血漿中活性型GIP[配列番号13]と非活性型GIP[配列番号14]の同時定量を行った。
1.試料の調製
(1)検量線用の試料の調製
実施例9と同様にして、表9のように試料を調製した。
α-グルコシダーゼ阻害剤を服用中の患者からたんぱく安定化剤入り採血管(べクトン・ディッキンソンアンドカンパニー製)を用いて採血し、血漿を得た。この血漿200μLに上記検量線作成時と同量の活性型GIP用内標準物質及び非活性型GIP用内標準物質を添加した。
2.測定
実施例8と同様にして、前処理を施した試料を測定した。測定条件は、表10~表12に示す。
糖尿病患者血漿試料を測定し、活性型GIP由来のペプチド断片[配列番号1]と活性型GIP用内標準物質由来のペプチド断片(アミノ酸配列は、配列番号1に同じ)をそれぞれ独立したピークとして検出できた(図16)。また、非活性型GIPも同様にそれぞれ独立したピークとして検出できた(図17)。活性型GIP濃度と両者のピーク面積比(活性型GIP由来ペプチドのピーク面積/内標準物質由来ペプチドのピーク面積)を線形最小二乗法により直線回帰して1pM~500pMの範囲で重み付け1/xを設定して検量線を作成した。糖尿病患者血漿試料から得られたピーク面積比(活性型GIP由来ペプチドのピーク面積/内標準物質由来ペプチドのピーク面積)をこの検量線に当てはめ、血漿中活性型GIP濃度を算出した。非活性型GIPは、10pM~500pMの範囲で重み付け1/xを設定して、活性型GIPと同様に検量線を作成し、糖尿病患者の血漿中非活性型GIP濃度を算出した。その結果を表13に示す。表中の数値は、それぞれの平均血漿中濃度±標準偏差を示す。
Claims (17)
- 試料中に含まれるグルカゴン・セクレチンファミリーペプチドのアミノ酸配列中に存在するアスパラギン酸のペプチド結合を切断し、グルカゴン・セクレチンファミリーペプチドをペプチド断片にする切断工程;
前記切断工程で切断されたペプチド断片を液体クロマトグラフィーにより分離精製し、測定対象物質であるグルカゴン・セクレチンファミリーペプチドのN末端側のペプチド断片を選定する分離精製工程;
前記分離精製工程で分離精製されたペプチド断片を質量分析し、グルカゴン・セクレチンファミリーペプチドのN末端側のペプチド断片を製造する分析工程;
を含む、試料中に含まれるグルカゴン・セクレチンファミリーペプチドの分析方法。 - 前記切断工程において、配列特異的切断プロテアーゼ又は酸から選択される切断物質を用いてアスパラギン酸のペプチド結合を切断する、請求項1に記載の分析方法。
- 前記切断物質が、エンドペプチダーゼASP-N、ギ酸、酢酸、トリフルオロ酢酸、プロピオン酸、及びそれらの組み合わせからなる群から選択される、請求項2に記載の分析方法。
- 前記分離精製工程において、液体クロマトグラフィーが、流速50nL/min~50μL/minの液体クロマトグラフィーである、請求項1~3のいずれかに記載の分析方法。
- 前記グルカゴン・セクレチンファミリーペプチドが、グルコース依存性インスリン分泌刺激ポリペプチド、グルカゴン様ペプチド-1、グルカゴン様ペプチド-2、グルカゴン、及びそれらの類縁体からなる群から選択される、請求項1~4のいずれかに記載の分析方法。
- 前記分離精製工程において、プレカーサーイオンの質量選択により測定対象物質を選定する、請求項1~5のいずれかに記載の分析方法。
- さらに、前記分離精製工程において、前記ペプチド断片を固相抽出する工程を含む、請求項1~6のいずれかに記載の分析方法。
- 前記分離精製工程及び分析工程が、高速液体クロマトグラフィー/マススペクトロメトリー/マススペクトロメトリー/マススペクトロメトリーにより行われる、請求項1~7のいずれかに記載の分析方法。
- 請求項1~8のいずれかに記載の分析方法を用いて検量線を作成し、前記検量線を用いて、試料中のグルカゴン・セクレチンファミリーペプチドを定量する、試料中のグルカゴン・セクレチンファミリーペプチドの定量方法。
- 2種以上のグルカゴン・セクレチンファミリーペプチドのペプチド断片を、同時にそれぞれ測定することにより、試料中の2種以上のグルカゴン・セクレチンファミリーペプチドを区別して同時に定量する、請求項9に記載の定量方法。
- 活性型グルカゴン・セクレチンファミリーペプチドのペプチド断片と、非活性型グルカゴン・セクレチンファミリーペプチドのペプチド断片とを、同時にそれぞれ測定することにより、試料中のグルカゴン・セクレチンファミリーペプチドの活性型と非活性型とを区別して同時に定量する、請求項9又は10に記載の定量方法。
- 前記検量線を、安定同位体で標識された内標準物質を試料に加えて作成する、請求項9~11のいずれかに記載の定量方法。
- 測定対象のペプチド断片及び内標準物質のペプチド断片の各ピーク面積比を用いて定量する、請求項12に記載の定量方法。
- 前記内標準物質が、グルカゴン・セクレチンファミリーペプチドのアミノ酸配列において1位~15位から選択される1又はそれ以上のアミノ酸を、安定同位体で標識されたアミノ酸で置換したペプチドである、請求項12又は13に記載の定量方法。
- (a)コントロール用マトリクス、
(b)内標準物質又はその溶液、
(c)グルカゴン・セクレチンファミリーペプチド又はそれらの標準物質、若しくはそれらの溶液、
(d)切断物質、
(e)固相抽出プレート
を含む、グルカゴン・セクレチンファミリーペプチドの定量用キット。 - 前記切断物質が、エンドペプチダーゼASP-N、ギ酸、酢酸、トリフルオロ酢酸、プロピオン酸、及びそれらの組み合わせからなる群から選択される、請求項15に記載の定量用キット。
- 前記グルカゴン・セクレチンファミリーペプチドが、グルコース依存性インスリン分泌刺激ポリペプチド、グルカゴン様ペプチド-1、グルカゴン様ペプチド-2、グルカゴン、及びそれらの類縁体からなる群から選択される、請求項15又は16に記載の定量用キット。
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JP2014238338A (ja) * | 2013-06-07 | 2014-12-18 | 株式会社Lsiメディエンス | 質量分析計を用いた生体試料中の蛋白質測定法 |
WO2018124011A1 (ja) | 2016-12-26 | 2018-07-05 | 花王株式会社 | 運動調節機能向上剤 |
WO2018124009A1 (ja) | 2016-12-26 | 2018-07-05 | 花王株式会社 | 低体温改善剤 |
WO2018124010A1 (ja) | 2016-12-26 | 2018-07-05 | 花王株式会社 | 認知機能改善剤 |
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EA201891323A1 (ru) | 2015-12-23 | 2019-02-28 | Амген Инк. | Способ лечения или облегчения метаболических нарушений с использованием белков, связывающих рецептор желудочного ингибиторного пептида (gipr), в комбинации с агонистами glp-1 |
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US10665440B1 (en) * | 2018-11-19 | 2020-05-26 | Thermo Finnigan Llc | Methods for multiplexed MS-3 analyses of peptides |
CA3123979A1 (en) * | 2018-12-21 | 2020-06-25 | Jiangsu Hengrui Medicine Co., Ltd. | Bispecific protein |
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