JP7067758B2 - A method for measuring the amount of angiotensin peptide in a sample and an angiotensin peptide quantification kit. - Google Patents

Description

The present invention relates to a method for measuring the amount of angiotensin peptide in a sample and a quantification kit of angiotensin peptide.

The renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure in the living body, and among them, the angiotensin peptide is a hormone that plays a central role in this system. Angiotensin peptides are produced from angiotensinogen (AGT), which is mainly produced in the liver, by enzymes such as renin and angiotensin-converting enzyme (ACE). It has been pointed out that local and tissue RAS of organs such as the heart and kidney are controlled by a mechanism different from that of systemic RAS, but there are still many unclear points. In particular, regarding renal RAS, there are some hypotheses regarding the production site and mechanism of angiotensin peptide production, and the physiological significance is also being discussed. Therefore, accurate quantification of angiotensin peptides in organs such as kidneys and biological samples such as blood and urine is expected to be useful for elucidating the involvement of tissues in the pathophysiology of RAS.

Currently, angiotensin peptides are quantified by antibody-based immunoassays such as radioimmunoassay (RIA) (see, for example, Non-Patent Document 1), enzyme immunoassay (ELISA), and the like. Often.

Further, as another quantification method, a measurement method combining liquid chromatography (LC) and mass spectrometry (MS) (hereinafter, may be referred to as “LC / MS”) is used. Further, quantified values of angiotensin peptide in kidney, urine, plasma or white adipose tissue have been reported (see, for example, Non-Patent Document 2).

Further, Patent Document 1 discloses a method for measuring renin activity by measuring the amount of angiotensin peptide in plasma using LC / MS.
Further, Patent Document 2 discloses a method of quantifying the amount of angiotensin peptide in plasma when the production and degradation cascade of RAS angiotensin peptide reaches an equilibrium state by using LC / MS.

Japanese Unexamined Patent Publication No. 2016-0756898 Japanese Patent Publication No. 2015-518964

Navar LG et al., “Tubular Fluid Concentrations and Kidney Contents of Angiotensins I and II in Anesthetized Rats”, J. Am. Soc. Nephrol., Vol.5, p1153-1158, 1994. Ali Q et al., “Estimation of angiotensin peptides in biological samples by LC / MS method”, Anal Methods, vol.6, no.1, p215-222, 2014.

In the method of quantifying angiotensin peptide by immunoassay using an antibody, the quantified value varies depending on the report. Although the antibody recognizes a specific amino acid sequence, it has been pointed out that accurate quantification may be difficult due to the cross-reaction of the antibody because each angiotensin peptide has high amino acid sequence homology. In addition, it is difficult to quantify a plurality of kinds of angiotensin peptides at the same time by the method for quantifying angiotensin peptides by immunoassay.
In addition, the previous reports of quantifying angiotensin peptides using LC / MS also include relative quantification, and the quantified values differ depending on the report. In particular, it is only Non-Patent Document 2 that the angiotensin peptide in the kidney was quantified using kidney, urine, plasma or white adipose tissue as a sample and LC / MS. However, the quantified value is about 1000 times the quantified value by the conventional immunoassay, which is far from the quantified value.
Further, in Patent Documents 1 and 2, the angiotensin peptide is quantified using LC / MS, but the purpose is to measure the renin activity of plasma, or the angiotensin peptide produced as a result of incubation for at least 15 minutes or more. The purpose is to quantify. Therefore, neither is a method for quantifying angiotensin peptides physiologically and pathophysiologically in vivo, and verification as a quantification method is insufficient.

The present invention has been made in view of the above circumstances, and provides a method for measuring the amount of angiotensin peptide in a sample capable of accurately quantifying angiotensin peptide, and an angiotensin peptide quantification kit.

That is, the present invention includes the following aspects.
The method for measuring the amount of angiotensin peptide in a urine sample according to the first aspect of the present invention is
After mixing the isotope-labeled angiotensin peptide with the urine sample , urea, thiourea and dithiothreitol are added as denaturants, and a solution obtained by denaturing the protein in the urine sample is dropped into an organic solvent. By precipitating the protein in the urine sample and suspending the precipitate in an organic solvent containing a strong acid, the endogenous angiotensin peptide and the isotope-labeled angiotensin peptide among the precipitates contain the strong acid. After resolubilization in an organic solvent to be used, centrifugation is performed to obtain a supernatant containing the endogenous angiotensin peptide and the isotope-labeled angiotensin peptide, and the supernatant is subjected to a solid phase separation method and liquid chromatography. Step 1 of separating and purifying the endogenous angiotensin peptide and the isotope-labeled angiotensin peptide from the urine sample in order of
Step 2 of detecting and quantifying the purified endogenous angiotensin peptide and the isotope-labeled angiotensin peptide by mass spectrometry.
Equipped with
The quantification limit of the angiotensin peptide is 0.1 fmol / μL or less.
The angiotensin peptide may be at least one in the group consisting of angiotensin 1, 2, 3, 4, 1-7, 1-9, 1-12, angiotensin A and allamandin.
In the method according to the first aspect, the amounts of two or more kinds of angiotensin peptides may be measured at the same time.
In step 2, multiple reaction monitoring may be used for detection using a triple quadrupole mass spectrometer.
In the method according to the first aspect, two or more mass transitions may be detected and quantified by the multiple reaction monitoring.

The angiotensin peptide quantification kit according to the second aspect of the present invention comprises an isotope-labeled angiotensin peptide, urea, thiourea and dithiothreitol , an organic solvent, a strong acid, and a solid-phase extraction column, and is a urine sample. Is the target sample.
The angiotensin peptide quantification kit according to the second aspect may include two or more isotope-labeled angiotensin peptides.

According to the above aspect, it is possible to provide a method for measuring the amount of angiotensin peptide in a sample capable of accurately quantifying angiotensin peptide and a quantification kit for angiotensin peptide.

It is a graph which shows the calibration curve in Example 1. FIG. Specifically, it is a graph which shows the calibration curve of angiotensin 1 in a plasma sample. It is a graph which shows the calibration curve in Example 1. FIG. Specifically, it is a graph which shows the calibration curve of angiotensin 2 in a plasma sample. Normal mice (controls), kidney-specific megalin knockout mice Ndrg1 ^Cre ERT2, megalin lox / lox (hereinafter referred to as "NDRG1 mice") and ApoE ^Cre , megalin lox / lox (hereinafter referred to as "ApoE mice") in Example 1. It is a graph comparing the quantitative value of angiotensin 1 in the urine sample (referred to as). 3 is a graph comparing the quantitative values of angiotensin 2 in urine samples of normal mice (control), NDRG1 mice and ApoE mice in Example 1. 3 is a graph comparing the quantitative values of angiotensin 1-9 in urine samples of normal mice (control), NDRG1 mice and ApoE mice in Example 1. 3 is a graph comparing the quantitative values of angiotensin 1-7 in urine samples of normal mice (control), NDRG1 mice and ApoE mice in Example 1. 3 is a graph comparing the quantitative values of angiotensin 2 in plasma samples of normal mice (control), NDRG1 mice and ApoE mice in Example 1. 3 is a graph comparing the quantitative values of angiotensin 1 in kidney samples of normal mice (control), NDRG1 mice and ApoE mice in Example 1. 3 is a graph comparing the quantitative values of angiotensin 2 in kidney samples of normal mice (control), NDRG1 mice and ApoE mice in Example 1. 3 is a graph comparing the quantitative values of angiotensin 1-9 in kidney samples of normal mice (control) and NDRG1 mice to which angiotensinogen was administered or not administered in Example 2. 3 is a graph comparing the quantitative values of angiotensin 1-7 in kidney samples of normal mice (control) and NDRG1 mice to which angiotensinogen was administered or not administered in Example 2. 3 is a graph comparing the quantitative values of angiotensin 1-9 in kidney samples of normal mice (control) and ApoE mice to which angiotensinogen was administered or not administered in Example 2. 3 is a graph comparing the quantitative values of angiotensin 1-7 in kidney samples of normal mice (control) and ApoE mice to which angiotensinogen was administered or not administered in Example 2.

<< Method for measuring the amount of angiotensin peptide in a sample >>
The method for measuring the amount of angiotensin peptide in a sample according to an embodiment of the present invention is a method for measuring the amount of angiotensin peptide in a sample, and the isotope-labeled angiotensin peptide is mixed with the sample. Then, the step 1 of purifying the endogenous angiotensin peptide and the isotope-labeled angiotensin peptide from the sample, and the purified endogenous angiotensin peptide and the isotope-labeled angiotensin peptide were detected by mass analysis. The method comprises the step 2 for quantification, and the quantification limit of the angiotensin peptide is 0.1 fmol / μL or less.

According to the method of this embodiment, the angiotensin peptide can be accurately quantified.

In the method of the present embodiment, the angiotensin peptide to be analyzed may be an enzymatic degradation product of angiotensinogen, and is not particularly limited. Specific examples of the angiotensin peptide include, and are limited to, angiotensin 1, angiotensin 2, angiotensin 1-9, angiotensin 1-7, angiotensin 1-12, angiotensin 3, angiotensin 4, angiotensin A, and allamandin. Not done.
Above all, in the method of the present embodiment, the angiotensin peptide to be analyzed is preferably at least one of the group consisting of angiotensin 1, angiotensin 2, angiotensin 1-9, and angiotensin 1-7.

The amino acid sequences of angiotensin 1, angiotensin 2, angiotensin 1-9, and angiotensin 1-7 are as shown below.
Angiotensin 1: DRVYIHPFHL (SEQ ID NO: 1)
Angiotensin 2: DRVYIHPF (SEQ ID NO: 2)
Angiotensin 1-9: DRVYIHPFH (SEQ ID NO: 3)
Angiotensin 1-7: DRVYIHP (SEQ ID NO: 4)

As shown in the above amino acid sequence, each angiotensin peptide has high amino acid homology. Therefore, in the conventional immunoassay method using an antibody, accurate quantification is difficult due to the cross-reactivity of the antibody. In addition, it has been difficult to quantify a plurality of types of angiotensin peptides at the same time by an immunoassay method using a conventional antibody. On the other hand, in the method of the present embodiment, since the quantification is performed by mass spectrometry, each of these angiotensin peptides can be identified and accurately quantified by the difference in mass. Furthermore, it is also possible to quantify a plurality of angiotensin peptides at the same time.

Further, until now, in the method of quantifying angiotensin peptide using LC / MS, the quantified value is far different from that of the immunoassay method using an antibody. This may be because the enzyme activity at the sample preparation stage was not sufficiently suppressed, the sample was not sufficiently purified, and the accuracy of the quantification method by mass spectrometry was insufficient. Be done.
On the other hand, in the present invention, the angiotensin peptide can be absolutely quantified by using a preparation method and a purification method according to the type of sample shown later, and a highly accurate quantification method using a mass spectrometry method.
The details of steps 1 and 2 of the method of the present embodiment will be described below.

<Step 1>
[Mixing process]
First, the isotope-labeled angiotensin peptide is mixed with the sample. At this time, by mixing the isotope-labeled angiotensin peptide having a known concentration, the endogenous angiotensin peptide contained in the sample can be absolutely quantified by the mass spectrometry method described later. In addition, the recovery rate of the angiotensin peptide in the sample can be calculated by mixing the isotope-labeled angiotensin peptide before purifying the sample.
The sample used in the method of the present embodiment may be a biological sample obtained from an animal, cultured cells or cultured tissue and may contain angiotensin peptide. Specific examples of the sample include tissue extracts derived from tissues or cultured tissues extracted from animals, cell extracts derived from cultured cells, body fluids, and the like.

Examples of the tissue or cultured tissue include, but are not limited to, vascular endothelium, brain, liver, kidney and the like.
Examples of the cultured cells include cells derived from the above-mentioned tissues.

Examples of the body fluid include blood, serum, plasma, urine (for example, raw urine, urine storage, etc.), buffy coat, saliva, bile, cerebrospinal fluid, tears, sputum, mucus, sweat, bladder lavage fluid, and the like. , Not limited to these.

When analyzing whole-body RAS, it is preferable to use body fluids such as blood, serum, and plasma that circulate throughout the body as samples.
On the other hand, when analyzing the RAS of a tissue, a tissue extract derived from a tissue or a cultured tissue extracted from an animal, a cell extract derived from a cultured cell, or a body fluid derived from a specific tissue is used as a sample. It is preferable to do so.
Specifically, for example, in order to analyze the RAS of the kidney, urine (preferably raw urine) may be used as the body fluid derived from a specific tissue.
Further, for example, in order to analyze the RAS of the brain, cerebrospinal fluid may be used.
Further, for example, in order to analyze the RAS of the liver / biliary system, bile may be used.
Until now, it has been difficult to absolutely quantify angiotensin peptides in tissues in order to analyze the RAS of tissues. On the other hand, according to the method of the present embodiment, a tissue extract derived from the tissue or cultured tissue extracted from the above-mentioned test animal, a cell extract derived from cultured cells, or a body fluid derived from a specific tissue is used as a sample. By using a preparation method and a purification method according to the type of the sample, and a highly accurate quantification method using a mass spectrometry method, the angiotensin peptide in the tissue can be absolutely quantified.

In addition, the animal from which the sample is derived is preferably a mammal. Examples of mammals include, but are not limited to, humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, rats and the like.

The isotope-labeled angiotensin peptide used in the method of the present embodiment may have ¹³ C and ¹⁵ N isotope-labeled amino acid residues. Specific examples of the ¹³ C and ¹⁵ N isotope-labeled amino acid residues include, but are not limited to, valine, arginine, isoleucine, leucine, lysine, phenylalanine, proline and the like. In the isotope-labeled angiotensin peptide, one of the amino acid residues may be substituted with ¹³ C and ¹⁵ N isotope-labeled amino acid residues, or two or more thereof may be substituted.
Among them, in the isotope-labeled angiotensin peptide used in the method of the present embodiment, the carbon atom of the arginine residue second from the N-terminal was replaced with the ¹³ C isotope, and the nitrogen atom was replaced with the ¹⁵ N isotope. It is preferable that it is a thing. This isotope-labeled angiotensin peptide results in an increase in mass of about 10 Da compared to the native angiotensin peptide.

Further, in step 1, the sample contains a plurality of types of endogenous angiotensin peptides. Therefore, when quantifying a specific one of the plurality of types of endogenous angiotensin peptides contained in the sample, they are mixed. As the isotope-labeled angiotensin peptide to be quantified, one kind corresponding to the quantified endogenous angiotensin peptide may be mixed.
On the other hand, when quantifying two or more of the multiple types of endogenous angiotensin peptides contained in the sample, the isotope-labeled angiotensin peptide to be mixed is a mixture of two or more types corresponding to the quantified endogenous angiotensin peptide. do it. By mixing two or more types of isotope-labeled angiotensin peptides with a sample, the amount of two or more types of endogenous angiotensin peptides can be quantified at the same time by the mass spectrometry method described later.

[Sample preparation process]
Further, the sample may be appropriately prepared before the mixing step. The sample preparation method may be appropriately performed by using a known method according to the type of sample and the like.
For example, when the sample is a tissue extract derived from a tissue extracted from an animal and a tissue extract derived from a cultured tissue, and a cell extract derived from a cultured cell, a known method (Reference 1: Navar LG et al., “Tubular Fluid Concentrations and” Kidney Contents of Angiotensins I and II in Anesthetized Rats ”, J. Am. Soc. Nephrol., Vol.5, p1153-1158, 1994.).
Specifically, first, the collected tissue, cultured cells, or cultured tissue is immediately homogenized in methanol which has been previously cooled to −80 ° C. The isotope-labeled angiotensin peptide is mixed with the obtained homogenate. Then, in order to remove excess protein, cell membrane, etc., centrifugation or the like is performed to obtain a supernatant thereof. Since this supernatant contains methanol, it is appropriately heated to evaporate the methanol. The obtained dried product can be dissolved in a sodium phosphate buffer solution or the like and used as a sample.

The above-mentioned method for preparing a tissue extract or a cell extract has been used so far when quantifying angiotensin peptide by RIA. However, for the first time, the present inventors have conducted a tissue extract or a cell extract (particularly, a kidney-derived tissue extract, as shown in Examples described later) when quantifying the angiotensin peptide by mass spectrometry. When the preparation method was applied, the angiotensin peptide could be absolutely quantified. This is because the activity of enzymes involved in RAS can be sufficiently suppressed by immediately holding the tissue, cultured tissue, and cultured cells in methanol at a low temperature of -80 ° C, and angiotensin in vivo. It is presumed that this is because the amount of peptide can be accurately quantified.

When the sample is a body fluid containing an enzyme such as protease (for example, blood, serum, plasma, raw urine, etc.), it is preferable to add a protease inhibitor immediately after collecting the body fluid such as blood. As a result, the activity of the enzyme involved in RAS can be sufficiently suppressed, and the amount of angiotensin peptide in the living body can be accurately quantified.
Specific examples of the protease inhibitor include metal protease inhibitors such as EDTA; angiotensin-converting enzyme (ACE) inhibitors such as captopril and lisinopril; and renin inhibitors such as aliskiren. Not limited to. These protease inhibitors may be added alone or in combination of two or more. Above all, in order to sufficiently suppress the activity of the enzyme involved in RAS, it is preferable to add a plurality of protease inhibitors in combination.
Further, after adding the protease inhibitor, the isotope-labeled angiotensin peptide may be mixed with a body fluid such as blood. When serum, plasma, or the like is used as a sample, a known method may be used to prepare the isotope-labeled angiotensin peptide from the mixed blood by adding a protease inhibitor.

[Refining process]
Then, the endogenous angiotensin peptide and the isotope-labeled angiotensin peptide contained in the sample are purified from the sample mixed with the isotope-labeled angiotensin peptide.
As the purification method, a known method may be appropriately selected depending on the type of sample.

Specific examples of the purification method include, and are not limited to, liquid chromatography (LC), solid-phase extraction method, and the like. Above all, it is preferable to carry out the solid-phase extraction method and LC in this order for separation and purification.

For example, when the sample is a body fluid, it is preferable to separate and purify by a peptide extraction method in order to reduce the protein in the sample before solid-phase extraction and separation and purification by LC.
Examples of the peptide extraction method include an albumin removal treatment method, an acetonitrile precipitation method, an ultrafiltration method, a DS method (Differential Solution method) and the like. Above all, it is preferable to use the DS method as the peptide extraction method.
So far, the DS method has been used for searching and quantifying biomarkers (excluding angiotensin peptides) by LC / MS using body fluids such as plasma as a sample. However, when the DS method was applied as a method for separating and purifying body fluids such as plasma and urine when quantifying angiotensin peptide by mass spectrometry for the first time, the present inventors applied a trace amount of several tens of μL or the like. The absolute quantification of the angiotensin peptide contained in the sample could be performed. It is presumed that this is because the angiotensin peptide was obtained from the sample with a higher recovery rate by using the DS method.
In particular, when separating and purifying a body fluid sample having a protein content lower than that of plasma (for example, urine as shown in Examples described later), it is common to apply an ultrafiltration method. .. On the other hand, it was discovered for the first time by the present inventors that a high recovery rate can be achieved by separating and purifying a body fluid sample containing almost no protein such as urine by using the DS method. Is.
That is, in the method of the present embodiment, when the sample is a body fluid, it is preferable to carry out the DS method, the solid-phase extraction method, and the LC in this order for separation and purification.

Specifically, the sample processing method using the DS method may be performed by the procedure shown below.
First, a denaturing agent is added to the body fluid.
Examples of the denaturing agent include, but are not limited to, urea, thiourea, dithiothreitol (DTT) and the like. These denaturants may be used alone or in combination of two or more. Above all, it is preferable to use urea, thiourea, and DTT in combination because the protein can be denatured more effectively.

Then, the body fluid to which the denaturant is added is slowly added dropwise to an organic solvent such as acetone cooled to about 4 ° C. After the dropping, the mixture is mixed at about 4 ° C. for about 30 minutes or more and about 3 hours or less to denature the protein in the sample and precipitate it. Then, it is separated by centrifugation or the like, and the supernatant is removed to obtain a precipitate. The obtained precipitate is suspended in a strong acid such as hydrochloric acid and an organic solvent such as acetonitrile. After suspension, the mixture is mixed at about 4 ° C. for about 30 minutes or more and about 3 hours or less to resolubilize the endogenous angiotensin peptide and the isotope-labeled angiotensin peptide among the denatured proteins. Then, the endogenous angiotensin peptide and the isotope-labeled angiotensin peptide can be separated and purified by separating them by centrifugation or the like to obtain a supernatant. At this time, since acetonitrile is contained in the supernatant, acetonitrile is evaporated by drying if necessary. The obtained dried product can be dissolved in an appropriate buffer solution to prepare a sample used for solid-phase extraction or separation and purification by LC.

(Liquid Chromatography (LC))
In addition, "liquid chromatography (LC)" in the present specification means that one or more components of a fluid solution are selectively selected as the fluid permeates uniformly through a column of a pulverized substance or a capillary channel. Means a delayed process. This delay results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid (ie, mobile phase) as the fluid moves relative to the stationary phase.
Specifically, as liquid chromatography, for example, reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC), turbulent liquid chromatography (TFLC) (high turbulent liquid chromatography (HTLC) or high throughput). (Also also referred to as liquid chromatography) and the like.

In separation and purification by liquid chromatography (LC), known LC devices and columns can be appropriately selected depending on the type of angiotensin peptide. The chromatographic column typically comprises a medium (ie, a packing component) and facilitates separation (ie, fractionation) of the chemical moiety. The medium may contain fine particles.
Specifically, as a separation and purification method by LC, for example, a sample (or a pre-purified sample) is introduced into a column from an inflow port. The sample can then be eluted with a solvent or solvent mixture and discharged from the outflow port to give the resulting sample.
Also, different elution modes may be selected to elute the angiotensin peptide. Elution modes of liquid chromatography include, for example, gradient mode, isotonic mode, polymorphic (ie, mixed) mode and the like.
During chromatography, separation of components (andiotensin peptides in this embodiment) is performed under various conditions such as eluent (also referred to as "mobile phase"), elution mode, gradient conditions, temperature and the like.

(Solid-phase extraction method)
In the present specification, solid phase extraction (SPE) is one of the analytical chemistry methods, in which the target compound and impurities in a solution or suspension are based on physical or chemical properties. Means how to separate. For example, as the mobile phase passes through or around the solid phase, the undesired components of the mobile phase are retained by the solid phase and the desired compound in the mobile phase is purified. Also, for example, the compound of interest in the sample may be retained by the solid phase, allowing unwanted components of the mobile phase to pass through or around the solid phase. In this case, the second mobile phase may then be used to elute the retained compound of interest from the solid phase for further treatment or analysis.
As the solid-phase extraction column, a commercially available one may be used, and examples thereof include Mono Spin (registered trademark) series (manufactured by GL Science).

<Step 2>
Then, it is introduced into a mass spectrometer after separation and purification to detect and quantify the endogenous angiotensin peptide and the isotope-labeled angiotensin peptide.

As used herein, "Mass Spectrometry (MS)" means an analytical technique for ionizing a molecule and identifying a compound by its mass.
As a mass spectrometry method, specifically, as shown below, an ionization step, a detection step, and a quantification step are performed in this order based on the mass-to-charge ratio (m / z) of the molecule.

[Ionization process]
In the method of this embodiment, the analysis target needs to be ionized by the mass spectrometer. Therefore, the endogenous angiotensin peptide and the isotope-labeled angiotensin peptide contained in the sample after separation and purification are ionized before being introduced into the mass spectrometer.

Methods for ionizing endogenous angiotensin peptides and isotope-labeled angiotensin peptides include, for example, electron ionization, chemical ionization, electrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization (APCI), photoionization, and atmospheric pressure photoionization. (APPI), Fast Atomic Impact (FAB), Liquid Secondary Ionization (LSI), Matrix Assisted Laser Desorption Ionization (MALDI), Electricity Ionization, Electricity Desorption, Thermospray / Plasma Spray Ionization, Surface Enhanced Laser Desorption Ionization (APPI) SELDI), induced coupled plasma (ICP), particle beam ionization and the like.
The ionization method can be appropriately selected based on the type of angiotensin peptide, the type of sample, the type of detector, the selection of the positive / negative mode, and the like.

Among them, as a method for ionizing an endogenous angiotensin peptide and an isotope-labeled angiotensin peptide, electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) is preferably used, and electrospray ionization (ESI) is used in the positive mode. ) Is more preferable.

[Detection process]
The amount of ions of the endogenous angiotensin peptide and the isotope-labeled angiotensin peptide is then detected.
Specifically, after the sample is ionized, the positively charged or loaded electric ions generated by it can be analyzed to determine the mass-to-charge ratio. Examples of the analyzer for determining the mass-to-charge ratio include a triple quadrupole mass analyzer, a quadrupole mass analyzer, an ion trap analyzer, a Fourier transform analyzer, an orbittrap analyzer, and a flight time analysis. Examples include vessels.
Ions can be detected using several detection modes. Specifically, for example, ions are detected using highly selective reaction monitoring (H-SRM), multiple reaction monitoring (MRM), selective reaction monitoring (SRM), selective ion monitoring mode (SIM), and the like. May be done.

Above all, the mass-to-charge ratio of the ions of the endogenous angiotensin peptide and the isotope-labeled angiotensin peptide is preferably detected using a triple quadrupole mass analyzer and multiple reaction monitoring.
Furthermore, it is preferable to detect two or more mass transitions when using multiple reaction monitoring, as it can be quantified more accurately.
As a result, as shown in the quantification step described later, two or more sub-peak areas are obtained from mass plots obtained from two or more mass transitions, and two or more quantified values are further obtained from two or more sub-peak areas. By calculating the above and calculating the average value of those quantitative values, a more accurate quantitative value of the angiotensin peptide can be obtained.

The triple quadrupole mass spectrometer is also called a "tandem mass spectrometer (MS / MS)" and can improve the selectivity of MS technology. In this technique, precursor ions (also called parent ions) produced from the molecule of interest are filtered in an MS device, followed by fragmentation of the precursor ions to one or more product ions (daughter ions or fragments). Also called an ion), which is then analyzed in the second MS procedure. By selecting precursor ions, only the ions produced by a particular molecule (angiotensin peptide in this embodiment) pass through the fractionation chamber, where product ions are generated by collision with the atoms of the inert gas. do. Both precursor and product ions are reproducibly produced under a given set of ionization / fragmentation conditions. Therefore, MS / MS technology is a very powerful analytical tool. For example, a filtration / fractionation combination can be used to eliminate interfering substances, which is particularly useful for complex samples such as biological samples.

[Quantitative process]
In the quantification step, only when a daughter ion generated by dissociation of an ionized target peptide (parent ion, that is, angiotensin peptide) is detected, it is detected as a signal. From the peak of the signal, the desired angiotensin peptide can be quantified. Further, at this time, by adding a known amount of an isotope-labeled peptide to the angiotensin peptide to be quantified and using it as an internal standard, it becomes possible to calculate the recovery rate and the absolute quantification value.

More specifically as a quantification method, for example, the amount of ions data is relayed to a computer and generated as a plot of the number of ions vs. time. From this plotted mass chromatogram, the area under the peak of the endogenous angiotensin peptide (eg, angiotensin 1) and the isotope-labeled angiotensin peptide (eg, isotope-labeled angiotensin 1) can be determined. Using these areas, the amount of endogenous angiotensin peptide can be calculated from the following formula [1].
Concentration of angiotensin peptide in the sample = (area under the peak of the sample / area under the peak of the internal standard) x concentration of the internal standard ... [1]

Further, by mixing two or more kinds of isotope-labeled angiotensin peptides, the absolute amount of two or more kinds of angiotensin peptides can be obtained at the same time.

<Preferable Embodiment>
[Sample: Tissue extract or cell extract]
In the method of this embodiment, when the sample is a tissue extract or a cell extract, the preferred embodiment is as shown below.
First, a sample is prepared using the above-mentioned method for preparing a tissue extract or a cell extract, and an isotope-labeled angiotensin peptide is mixed. The mixture is then passed through a solid phase column and further separated and purified by liquid chromatography. The separated purified product is then introduced into a triple quadrupole mass spectrometer, electrospray ionized (ESI) in positive mode, and using multiple reaction monitoring, two of the endogenous angiotensin peptide and the isotope-labeled peptide. The above mass transition is detected. Mass chromatograms are generated from two or more mass transitions of the detected endogenous angiotensin peptide and isotope-labeled peptide to determine the area under each of the two or more peaks. Next, the quantitative values of the two or more endogenous angiotensin peptides are calculated from the areas under the two or more peaks of the endogenous angiotensin peptide and the isotope-labeled peptide using the above formula [1]. Next, the average of the quantitative values obtained from two or more mass transitions is taken as the quantitative value, and the final quantitative value is the average value when the same sample is measured twice or more.

[Sample: Body fluid]
In the method of this embodiment, when the sample is a body fluid, the preferred embodiment is as shown below.
First, the sample and the isotope-labeled angiotensin peptide are mixed. Then, it is separated and purified using the DS method. The mixture is then passed through a solid phase column and further separated and purified by liquid chromatography. The separated purified product is then introduced into a triple quadrupole mass spectrometer, electrospray ionized (ESI) in positive mode, and using multiple reaction monitoring, two of the endogenous angiotensin peptide and the isotope-labeled peptide. The above mass transition is detected. Mass chromatograms are generated from two or more mass transitions of the detected endogenous angiotensin peptide and isotope-labeled peptide to determine the area under each of the two or more peaks. Next, the quantitative values of the two or more endogenous angiotensin peptides are calculated from the areas under the two or more peaks of the endogenous angiotensin peptide and the isotope-labeled peptide using the above formula [1]. Next, the average value of two or more quantitative values is taken as the final quantitative value.

[Quantitative limit]
In the method of the present embodiment, the quantification limit (lower limit of quantification) of the angiotensin peptide is preferably 0.5 fmol / μL or less, and more preferably 0.1 fmol / μL or less.
On the other hand, the upper limit of quantification of the angiotensin peptide is, for example, 300 fmol / μL or more.

In addition, in this specification, "limit of quantation (LOQ)" means the point that measurement becomes quantitatively meaningful. The sample response at this limit of quantification can be identified, separated and reproduced with an accuracy of 20% and an accuracy of 80-120%. Further, the "quantitative upper limit" means a linear upper limit range in which the sample response can be quantified.

≪Angiotensin peptide quantification kit≫
The angiotensin peptide quantification kit according to an embodiment of the present invention comprises an isotope-labeled angiotensin peptide and a denaturing agent.

According to the quantification kit of the present embodiment, the angiotensin peptide can be accurately quantified.

Examples of the isotope-labeled angiotensin peptide included in the quantification kit of the present embodiment include those similar to those exemplified in the above-mentioned step 1. Among them, as the isotope-labeled angiotensin peptide included in the quantification kit of the present embodiment, the carbon atom of the second arginine residue from the N-terminal is replaced with the ¹³ C isotope, and the nitrogen atom is replaced with the ¹⁵ N isotope. It is preferable that it is an isotope. This isotope-labeled angiotensin peptide results in an increase in mass of about 10 Da compared to the native angiotensin peptide.

Further, the isotope-labeled angiotensin peptide included in the quantification kit of the present embodiment may be one kind or two or more kinds. By providing two or more types of isotope-labeled angiotensin peptides, the amount of two or more types of angiotensin peptides can be quantified at the same time.

Examples of the denaturing agent included in the quantitative kit of the present embodiment include the same as those exemplified in the above-mentioned step 1. Among them, the denaturing agent included in the quantitative kit of the present embodiment is preferably at least one of the group consisting of urea, thiourea, and DTT, and at least one of the group consisting of urea, thiourea, and DTT. Two are more preferred, and urea, thiourea, and DTT are even more preferred.

The quantitative kit of the present embodiment may further include a solid-phase extraction column or the like. By providing a solid-phase extraction column, the degree of purification of the sample before separation and purification by LC can be further improved.
Examples of the solid-phase extraction column include those similar to those exemplified in the above-mentioned step 1.

The quantitative kit of the present embodiment may further appropriately include a solvent, a reagent, a container and the like used for separation and purification by LC and mass spectrometry.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the following examples.

[Example 1]
1. 1. Sample preparation As subjects, normal male mice (C57BL / 6J) 12 to 15 weeks old (purchased from Charles Rivers Laboratories International), and megalin complete knockout (KO) mice (Ndrg1 ^Cre ERT2, megalin lox /), which are transgenic mice. lox) (hereinafter, may be referred to as “NDRG1 mouse”) and megalin partial KO mouse (ApoE ^Cre , megalin lox / lox) (hereinafter, may be referred to as “ApoE mouse”) were used.

(1) Collection of urine The urine stored for 24 hours before dissection was used as a sample. The obtained urine was subjected to low-speed centrifugation to remove cellular components and the like, and the supernatant was used. The resulting supernatant was frozen and stored at −80 ° C. until use.

(2) Blood collection After sedation of mice using exhaled anesthesia isoflurane, cervical spine dislocation was performed and a midline incision was made. Then, 0.8 mL of blood was collected from the inferior vena cava. At this time, 50 μL of 500 mM ethylenediaminetetraacetic acid (EDTA), 6.45 μL of 2 mM Captopril, 25 μL of synthetic protease inhibitor complete (1 tablet made by Rosche dissolved in 2 mL of ultrapure water), and 10 μL of 25 mM Aliskiren were previously filled per 1 mL of blood. Blood was immediately collected in a 1 mL syringe. The blood was then centrifuged at 800 xg for 20 minutes at room temperature, the supernatant plasma was collected and frozen in liquid nitrogen. The resulting plasma was stored at −80 ° C. until use. In the obtained plasma, we requested Oriental Yeast Co., Ltd. to confirm that the renin activity was sufficiently suppressed by the RIA2 antibody method. In addition, it was confirmed that the ACE activity was sufficiently suppressed by adding angiotensin 1 to plasma and measuring the amount of change to angiotensin 2.

(3) Kidney removal After blood collection, the right kidney was removed, the capsule was removed, cut in half, and immediately frozen in liquid nitrogen. The left kidney was also removed, treated in the same manner, and frozen. The resulting kidney was stored at −80 ° C. until use.

2. 2. Step 1: Purification of angiotensin peptide (1) Separation of angiotensin peptide from each sample-Separation of angiotensin peptide from urine sample First, 40 μL of urine with stable isotope-labeled angiotensin peptide (1, 2, 1-9 and 1) -7) (Angiotensin peptides 1 and 2 were manufactured by Graner bio-one, and angiotensin peptides 1-9 and 1-7 were manufactured by Sigma-Aldrich) were added by 100 fmol each. The amino acid sequence of each angiotensin peptide is as shown in SEQ ID NOs: 1 to 4. A double dose of the sample was then added (7M urea, 2M thiourea, and 20 mM dithiothreitol (DTT)). The mixture was then slowly added drop by drop into 2 mL of acetone cooled to 4 ° C. using a fine gel loading tip. Immediately after the addition, the mixture was mixed at 4 ° C. for 1 hour. Then, it was centrifuged at 4 ° C. and 19000 × g for 15 minutes, and the supernatant was discarded. The precipitate was then resuspended with a mixed solution of 1 mL of 12 mM hydrochloric acid (HCl) and 80% acetonitrile (ACN). Then, it was mixed at 4 ° C. for 2 hours. Then, it was centrifuged at 4 ° C. and 19000 × g for 15 minutes, and the supernatant was collected. Then, the collected supernatant was dried by a centrifugal dryer to obtain a dried product. The resulting dried product was resuspended in 1 mL of 0.2% trifluoroacetic acid (TFA).

-Separation of angiotensin peptide from plasma sample First, 100 fmol of stable isotope-labeled angiotensin peptide (1, 2, 1-7 and 1-9, manufactured by Sigma-Aldrich) was added to 20 μL of plasma. A double dose of the sample was then added (7M urea, 2M thiourea, and 20 mM dithiothreitol (DTT)). The mixture was then slowly added drop by drop into 2 mL of acetone cooled to 4 ° C. using a fine gel loading tip. Immediately after the addition, the mixture was mixed at 4 ° C. for 1 hour. Then, it was centrifuged at 4 ° C. and 19000 × g for 15 minutes, and the supernatant was discarded. The precipitate was then resuspended with a mixed solution of 800 μL of 12 mM hydrochloric acid (HCl) and 80% acetonitrile (ACN). Then, it was mixed at 4 ° C. for 1 hour. Then, it was centrifuged at 4 ° C. and 19000 × g for 15 minutes, and the supernatant was collected. Then, the collected supernatant was dried by a centrifugal dryer to obtain a dried product. The resulting dried product was resuspended in 1 mL of 0.2% TFA.

-Separation of angiotensin peptide from kidney sample First, after measuring the wet weight of the kidney, the right kidney cut in half was homogenized in 2.5 mL of methanol (pre-chilled at -80 ° C) (condition:). IKA homogenizer, 30 seconds). In the obtained renal homogenate, we requested Oriental Yeast Co., Ltd. to confirm that the renin activity was sufficiently suppressed by the RIA2 antibody method. In addition, it was confirmed that the ACE activity was sufficiently suppressed by adding angiotensin 1 to the obtained renal homogenate and measuring the amount of change to angiotensin 2.
Then, 500 fmol of each of stable isotope-labeled angiotensin peptides (1, 2, 1-7 and 1-9, manufactured by Sigma-Aldrich) was added to the obtained homogenate as an internal standard. Then, it was centrifuged at 4 ° C. and 1400 × g for 15 minutes, and the supernatant was collected in a 15 mL heat-resistant glass test tube. The test tube containing the supernatant was then heated with a heat block heated to 85 ° C. to evaporate the methanol. After drying, it was resuspended in 500 μL of Angiotensin assay buffer (containing 50 mM sodium-phosphate and 1 mM EDTA, pH 7.4). Then, it was centrifuged at 4 ° C. and 21000 × g for 20 minutes, and the supernatant was collected. Then, 500 μL of 0.2% TFA was added to the collected supernatant to make a total of about 1 mL.

(2) Desalination and peptide fractionation of each sample ・ Desalination and peptide fractionation of urine sample and kidney sample Next, the resuspension derived from urine and kidney obtained in (1) was transferred to Mono Spin (registered trademark). Desalination and peptide fractionation were performed using C18 (manufactured by GL Science). The specific procedure is as shown below.
First, a solvent or a sample was added dropwise to Mono Spin® C18 (manufactured by GL Science), and the mixture was centrifuged at 4 ° C. and 1000 × g for 1 minute. The order in which the solvent and the sample were dropped is as shown below.
(Column equilibration)
5 mL Methanol → 5 mL 0.2% TFA and 80% ACN mixture → 5 mL 0.2% TFA
(Dripping of sample)
→ Resuspension (washing) obtained in 1 mL (1)
→ 5mL 0.2% TFA
(Elution)
→ 2mL 0.2% TFA and 30% ACN mixture

Plasma sample desalting and peptide fractionation The plasma-derived resuspension obtained in (1) was then desalted and peptide fractionated using Mono Spin® C18 (manufactured by GL Science). The specific procedure is as shown below.
First, a solvent or a sample was added dropwise to Mono Spin® C18 (manufactured by GL Science), and the mixture was centrifuged at 4 ° C. and 1000 × g for 1 minute. The order in which the solvent and the sample were dropped is as shown below.
(Column equilibration)
5 mL Methanol → 5 mL 0.2% TFA and 80% ACN mixture → 5 mL 0.2% TFA
(Dripping of sample)
→ Resuspension (washing) obtained in 1 mL (1)
→ 5mL 0.2% TFA
→ 5mL 0.2% TFA and 15% ACN mixture (elution)
→ 2mL 0.2% TFA and 30% ACN mixture

(3) Final Preparation of Samples Each eluate containing the angiotensin peptide obtained by the above-mentioned treatment with Mono Spin C18 was dried in a centrifugal dryer. The kidney sample was then resuspended in 100 μL and the urine and blood samples were resuspended in 20 μL each with 0.2% TFA to prepare the samples for liquid chromatography.

(4) Separation and Purification by Liquid Chromatography Separation and Purification of Urine and Kidney Samples by Liquid Chromatography As a liquid chromatography (LC) system, Exgent (registered trademark) Expert nano LC 400, Exgent (registered trademark). Expert cHiPLC® was used. The column temperature was set to 30 ° C. and used in the Trap and elute mode.

The columns, mobile phases and flow velocities used are as shown below.
(column)
Trap volume: Nano cHiPLC® Trap volume 200 μm × 0.5 mm ChromXP C18-CL 3 μm 120 Å (804-00006)
Analytical volume: Nano cHiPLC® volume 75 μm × 15 cm ChromXP C18-CL 3 μm 120 Å (804-00001)
(Mobile phase)
Solvent A: 0.1% formic acid (FA)
Solvent B: 0.1% FA and 100% ACN mixed solution (flow velocity)
300nL / min

The procedure for separation and purification by LC is as shown below.
The time for introducing the sample finally prepared in (3) into the Analytical volume equilibrated with 98% of A solvent and 2% of B solvent was set to 0 minutes. First, the solvent B was linearly increased to 32% in 30 minutes, and the angiotensin peptide to be measured was separated and eluted. Then, in 5 minutes from there, the B solvent was raised to 90%. Then, the column was washed with 10% of A solvent and 90% of B solvent for 5 minutes. Then, it was returned to 98% of A solvent and 2% of B solvent in 0.1 minute, and equilibrated with 98% of A solvent and 2% of B solvent in the initial state until 60 minutes.

-Separation and purification of plasma samples by liquid chromatography The LC system, column, mobile phase and flow velocity used are the same as those of urine and kidney samples by liquid chromatography.
The procedure for separation and purification by LC is as shown below.
The time for introducing the sample finally prepared in (3) into the Analytical volume equilibrated with 90% of A solvent and 10% of B solvent was set to 0 minutes. First, the solvent B was linearly increased to 28% in 30 minutes, and the angiotensin peptide to be measured was separated and eluted. Then, in 5 minutes from there, the B solvent was raised to 90%. Then, the column was washed with 10% of A solvent and 90% of B solvent for 5 minutes. Then, it was returned to 98% of A solvent and 2% of B solvent in 0.1 minute, and equilibrated with 98% of A solvent and 2% of B solvent until 70 minutes.

3. 3. Step 2: Mass spectrometry (ionization, detection and quantification of angiotensin peptide)
For the mass spectrometer, Triple TOF® 5600+ manufactured by Siex was used in the Positive ion mode.

-Method of analyzing urine samples Mass spectrometry of Angiotensin I, II, 1-9 and 1-7 using urine samples was performed under the following conditions.
(Conditions for mass spectrometry)
TOF MS mass range: 200-1200 Da
Declustering positive (DP): 80V
Collision energy (CE): 10V
TOF-MS accumulation time: 0.25 seconds Production ion accumulation time: 0.199 seconds Cycle time: 1.89 seconds Total 1895 cycles The conditions for the collision-induced dissociation reaction are optimized using the standard products of each angiotensin peptide. Was done.
The conditions for acquiring the MS / MS spectrum of each angiotensin peptide are as shown in Table 1 below.

-Analysis method of plasma sample and kidney sample Mass spectrometry of Angiotensin I and II using plasma sample and kidney sample was performed under the following conditions.
(Conditions for mass spectrometry)
TOF MS mass range: 400-1250 Da
Declustering positive (DP): 80V
Collision energy (CE): 10V
TOF-MS accumulation time: 0.25 seconds Production ion accumulation time: 0.20 seconds Cycle time: 1.1 seconds Total 3272 cycles The conditions for the collision-induced dissociation reaction are optimized using standard products of each angiotensin peptide. Was done.
The conditions for acquiring the MS / MS spectrum of each angiotensin peptide are as shown in Table 2 below.

For each sample, the generated ions are passed through a first quadrupole (Q1) set to select precursor ions whose mass-to-charge ratio is the value shown in Table 3. rice field. Then, the ions flowing into the second quadrupole were made to collide with the argon gas to generate an ion fraction. Next, from each ion fraction obtained in Q2, a third quadrupole (Q3) set to select a product ion (Production) whose mass-to-charge ratio is the value shown in Table 3. Was passed through. Further, as shown in Table 3, angiotensin 1, 1-9 and 1-7 form three mass transitions and angiotensin 2 has two mass transitions, which are a combination of precursor ions and product ions. I made a mass transition. As the quantitative value of the angiotensin peptide in each sample, the average value of the quantitative values obtained from these two or three mass transitions was used.
At the same time, for the stable isotope-labeled angiotensin peptide, which is an internal standard, the precursor ion and the product ion whose mass-to-charge ratio is the value shown in Table 3 are selected in the same manner. Mass spectrometry was performed.
In Table 3, "light" indicates an unlabeled angiotensin peptide, and "heavy" indicates a stable isotope-labeled angiotensin peptide.

Analyst® TF1.6 (manufactured by AB SCIEX) was used as the LC-MS / MS data acquisition and analysis software for each sample. Furthermore, Skyline (MacCoss Lab Software, University of Washington) was used as software for constructing a quantitative method and calculating a peak area value in the MRM-HR (registered trademark) method.
The "MRM-HR (registered trademark) method" is a method having almost the same principle as selective reaction monitoring (SRM). Specifically, in the MRM-HR (registered trademark) method, first, an ion of a peptide (target peptide) to be quantified in advance is selected as a Precursor ion, and only the selected Precursor ion is called a collision-induced dissociation method (CID). The method is a method in which a fragmentation is caused mainly by a peptide bond, and a product ion derived from the precursor ion is selectively detected.
As a concrete data analysis, first, a plot of the number of ions vs. time was generated. The area under the peak was measured, and the following formula [1] was calculated based on the concentration of the internal standard stabilized isotope-labeled angiotensin peptide (25 fmol / on volume (5 μL) for all urine, plasma, and kidney samples). Was used to quantify the concentration of the sample.
Concentration of angiotensin peptide in sample [fmol]
= (Area under the peak of the sample / Area under the peak of the internal standard) × Concentration of the internal standard ... [1]

In addition, a calibration curve was prepared using the following method, and since it contained an endogenous angiotensin peptide, only the slope of a straight line was used to calculate the quantitative value.
First, a matrix containing no stable isotope-labeled angiotensin peptide was prepared, a certain amount of stable isotope-labeled angiotensin peptide (heavy peptides) was added as an internal standard, and then the concentration of unlabeled angiotensin peptide (light peptides) was added. Addition was made to make a standard solution. As each unlabeled angiotensin peptide (1, 2, 1-9 and 1-7), a product manufactured by Sigma-Aldrich was used. heavy peptides were added so as to be 25 fmol / on collagen. In addition, "on volume" means introduction amount, and in this case, it is 5 μL. In addition, light peptides were added so as to be 0, 1, 2.5, 10, 50, 75, and 100 fmol / on volume.
Next, the ratio of the peak area value of light peptides and the peak area value of heavy peptides was taken (light / havey), the horizontal axis was the concentration of light peptides, and the vertical axis was the concentration of light peptides. .. The measurement was performed three times for each concentration standard solution. The calibration curve was created using the method of least squares, and the linearity of the calibration curve was evaluated.

In addition, the standard deviation (SD), accuracy and accuracy of the three measurements were calculated and the LLOQ (lower limit of quantification) was evaluated. The evaluation item of LLOQ was "satisfying precision ≤ 20% and accuracy ≤ 20%".
In addition, as a quality control sample, a standard solution having a low concentration (2.5 fmol / on volume), a medium concentration (50 fmol / on volume), and a high concentration (75 fmol / on volume) was measured 5 times each, and the precision and accuracy were measured. evaluated. The evaluation items were "satisfying precision ≤ 15% and accuracy ≤ 15%".
The calibration curves of angiotensin 1 and 2 in plasma samples are shown in FIGS. 1A (angiotensin 1) and FIG. 1B (angiotensin 2). In FIGS. 1A and 1B, "1st" means the first measured value, "2nd" means the second measured value, and "3rd" means the third measured value.

From FIGS. 1A (angiotensin 1) and 1B (angiotensin 2), the LLOQ satisfying precision ≦ 20% and accuracy ≦ 20% is 0.5 fmol / on volume for angiotensin 1 (AngI) and 0 for angiotensin 2 (AngII). It was .5 fmol / on volume.
Further, as a quality control sample, a standard solution having a low concentration (2.5 fmol / on volume), a medium concentration (50 fmol / on volume), and a high concentration (75 fmol / on volume) was measured 5 times each, and as a result, precision ≦ 15%. And it was confirmed that accuracy ≦ 15% was satisfied.

The quantitative results of the angiotensin peptide contained in each sample are shown in Table 4 below. In addition, the results of comparing the angiotensin peptides contained in each sample between normal mice, megalin complete knockout mice (NDRG1 mice) and megalin partial knockout mice (ApoE mice) are shown in FIGS. 2A (angiotensin 1 in urine) and 2B (urine). Angiotensin 2), FIG. 2C (angiotensin 1-9 in urine), FIG. 2D (angiotensin 1-7 in urine), FIG. 3 (angiotensin 1 in plasma), FIG. 4A (angiotensin 1 in kidney) and FIG. 4B (angiotensin 2 in the kidney) is shown. The concentration of angiotensin peptide in urine and plasma was shown in fmol / mL, and the concentration of angiotensin peptide in kidney was shown in fmol / g. Further, in the table and each figure, "NDRG1" is the measurement result of the NDRG1 mouse, "ConN" is the measurement result of the control (normal) mouse used as the control of the NDRG1 mouse, and "ApoE" is the measurement result of the ApoE mouse, "ConE". Shows the measurement results of the control (normal) mouse as a control of the ApoE mouse.

From Table 4 and FIGS. 2A to 2D, all of angiotensin 1, 2, 1-9 and 1-7 could be detected from the urine sample. In addition, it was clarified that the content of each angiotensin peptide was higher in NDRG1 mice and ApoE mice (particularly, NDRG1 mice) as compared with normal mice.
In the plasma sample, angiotensin peptides 1, 1-9 and 1-7 were below the lower limit of quantification, but angiotensin 2 could be detected. In addition, it was clarified that there was no significant difference in the content of angiotensin 2 between normal mice, NDRG1 mice and ApoE mice.
In the kidney sample, angiotensin peptides 1-9 and 1-7 were below the lower limit of quantification, but angiotensin 1 and 2 could be detected. In addition, it was clarified that there was no significant difference in the content of angiotensin 2 between normal mice, NDRG1 mice and ApoE mice.

[Example 2]
1. 1. Sample preparation As subjects, normal male mice (C57BL / 6J) 12 to 15 weeks old (purchased from Charles Rivers Laboratories International), transgenic mice, Megalin complete KO mice (NDRG1 mice) and Megalin partial KO mice (ApoE). Mouse) was used.

(1) Resection of the kidney 200 μL of 5 mg / mL mouse recombinant angiotensinogen (AGT) (that is, containing 1 mg of AGT) was intraperitoneally injected into a part of normal mice (control), NDRG1 mouse and ApoE mouse. Thirty minutes later, cervical spine dislocation was performed and a midline incision was made.
The right kidney was then removed, the capsule removed, cut in half, and immediately frozen in liquid nitrogen. The left kidney was also removed, treated in the same manner, and frozen. The resulting kidney was stored at −80 ° C. until use.

2. 2. Step 1: Purification of Angiotensin Peptide (1) Separation of Angiotensin Peptide from Kidney Samples Except for using only stable isotope-labeled angiotensins 1-9 and 1-7 as internal standards, the "kidney" of Example 1 (1). The angiotensin peptide was separated using the same method as in "Separation of angiotensin peptide from sample".

(2) Desalting and peptide fractionation of kidney sample Using the same method as in "Desalting and peptide fractionation of urine sample and kidney sample" in (1) of Example 1, desalting and peptide fractionation were performed.

(3) Separation and purification of kidney sample by LC Angiotensin peptide was separated and eluted using the same method as in "Separation and purification of urine sample and kidney sample by liquid chromatography" in Example 1 (3).

3. 3. Step 2: Mass spectrometry (ionization, detection and quantification of angiotensin peptide)
Using the same method with reference to the conditions of "Analysis method of urine sample" in "3. Steps 2 to 4: Mass spectrometry (ionization, detection and quantification of angiotensin peptide)" of Example 1, each mouse Angiotensin 1-9 and 1-7 contained in the kidney sample were mass-analyzed and quantified. The results are shown in Tables 5 and 6 below. In addition, the results of comparing the angiotensin peptides contained in the kidney sample between the normal mouse, the megalin complete knockout mouse (NDRG1 mouse) and the megalin partial knockout mouse (ApoE mouse) are shown in FIG. 5A (normal mouse and NDRG1 mouse angiotensin 1-9). 5B (normal mouse and NDRG1 mouse angiotensin 1-7), FIG. 6A (normal mouse and ApoE mouse angiotensin 1-9) and FIG. 6B (normal mouse and ApoE mouse angiotensin 1-9).
In Tables 5, 5A and 5B, "con (dis)" indicates the measurement result in a normal mouse to which AGT was not administered, and "con AGT + (dis)" is a measurement in a normal mouse to which AGT was administered. The results are shown, and "NDRG1 (dis)" indicates the measurement results in NDRG1 mice not administered with AGT, and "NDRG1 AGT + (dis)" indicates the measurement results in NDRG1 mice administered with AGT.
Further, in Tables 6, 6A and 6B, "con (dis)" indicates the measurement result in a normal mouse to which AGT was not administered, and "con AGT + (dis)" is a measurement in a normal mouse to which AGT was administered. The results are shown, and "ApoE (dis)" indicates the measurement results in ApoE mice not administered with AGT, and "ApoE AGT + (dis)" indicates the measurement results in ApoE mice administered with AGT.

From Table 5, FIGS. 5A and 5B, it was confirmed that administration of AGT increased the values of angiotensin 1-9 and 1-7 in the kidney sample in both normal mice and NDRG1 mice.
In addition, from Table 6, FIGS. 6A and 6B, it was confirmed that administration of AGT increased the values of angiotensin 1-9 and 1-7 in the kidney sample in both normal mice and ApoE mice. .. On the other hand, in normal mice and ApoE mice not administered with AGT, angiotensins 1-9 and 1-7 were below the lower limit of quantification.

According to the method and quantification kit of the present embodiment, the angiotensin peptide can be accurately quantified.

Claims (7)

A method for measuring the amount of angiotensin peptide in a urine sample.
After mixing the isotope-labeled angiotensin peptide with the urine sample , urea, thiourea and dithiothreitol are added as denaturants, and a solution obtained by denaturing the protein in the urine sample is dropped into an organic solvent. By precipitating the protein in the urine sample and suspending the precipitate in an organic solvent containing a strong acid, the endogenous angiotensin peptide and the isotope-labeled angiotensin peptide among the precipitates contain the strong acid. After resolubilization in an organic solvent to be used, centrifugation is performed to obtain a supernatant containing the endogenous angiotensin peptide and the isotope-labeled angiotensin peptide, and the supernatant is subjected to a solid phase separation method and liquid chromatography. Step 1 of separating and purifying the endogenous angiotensin peptide and the isotope-labeled angiotensin peptide from the urine sample in order of
Step 2 of detecting and quantifying the purified endogenous angiotensin peptide and the isotope-labeled angiotensin peptide by mass spectrometry.
Equipped with
A method in which the quantification limit of angiotensin peptide is 0.1 fmol / μL or less. The method according to claim 1 , wherein the angiotensin peptide is at least one in the group consisting of angiotensin 1, 2, 3, 4, 1-7, 1-9, 1-12, angiotensin A and allamandin. The method according to claim 2 , wherein the amounts of two or more kinds of angiotensin peptides are measured at the same time. The method according to any one of claims 1 to 3 , wherein in the step 2, a triple quadrupole mass spectrometer is used and multiple reaction monitoring is used for detection. The method according to claim 4 , wherein two or more mass transitions are detected and quantified by the multiple reaction monitoring. Isotope-labeled angiotensin peptide and
With urea, thiourea and dithiothreitol ,
With organic solvent
With strong acid,
Solid-phase extraction column and
Equipped with
Angiotensin peptide quantification kit for urine samples. The angiotensin peptide quantification kit according to claim 6 , which comprises two or more isotope-labeled angiotensin peptides.

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