JP5144273B2 - Polypeptide detection or quantification method and apparatus - Google Patents

Description

  The present invention relates to a highly sensitive polypeptide detection or quantification method and apparatus using reversed-phase liquid chromatography.

  Reverse-phase liquid chromatography (RP-LC) is liquid chromatography that uses a substance having a polarity less than that of a mobile phase as a stationary phase, and is most widely used for quantifying low-molecular compounds such as drugs. In recent years, reverse phase liquid chromatography has been used as an important technique in the detection and quantification of polypeptides, and also plays an important role in proteomics research.

  One of the major problems that makes it difficult to perform high-sensitivity quantification of polypeptides by reversed-phase liquid chromatography is polypeptide adsorption. Polypeptide adsorption becomes more intense at low concentrations (Non-Patent Documents 1 to 3), and as a result, it is considered that quantification in a low concentration region is difficult. In order to solve this adsorption at a low concentration, it has been shown that addition of a polypeptide such as albumin or a surfactant (Non-patent Document 1) is effective in suppressing these adsorptions. However, for each polypeptide to be measured, in addition to the need to confirm the anti-adsorption effect by addition, it is confirmed that the added surfactant does not cause problems such as appearing as a contaminated peak during measurement. There is a need. In general, introduction of large amounts of polypeptide into a reverse phase liquid chromatograph contributes to clogging of the column. In addition, when the volume of the polypeptide sample exceeds the column volume, it is difficult to sufficiently retain the test polypeptide on the column and to separate it, resulting in loss of reproducibility and difficulty in quantification.

On the other hand, if a large amount of surfactant is present at the time of analysis, the column may be deteriorated and the instrument such as mass spectrometry may be contaminated and reproducibility may be lost. There are cases where this is difficult. For this reason, a system for removing surfactants online using the column switching method has also been devised, but it requires extremely complicated studies such as the effect of the surfactant must be confirmed for each polypeptide. .
J. et al. Mass Spectrom. 1998, 33, 967-983 Anal. Biochem. 1994, 222, 149-155 J. et al. Chromatogr. B 2002,775,247-255 Pharmaceutical Research 2000, 17, 1551-1557.

  An object of the present invention is to provide a method and apparatus for detecting or quantifying a polypeptide by reversed-phase liquid chromatography that eliminates the influence of polypeptide adsorption by a simple technique and enables accurate quantification of the polypeptide even in a low concentration region. Is to provide.

  The present inventors have intensively studied to solve the above problems, and the ability of the polypeptide dissolved in a mixed solution consisting essentially of water and one kind of organic solvent to adsorb to the reversed-phase column filler is the mixed solution. Depends on the volume ratio (%) of the organic solvent at a constant volume ratio (referred to simply as the phase transition critical value of the adsorption ability of the polypeptide to the reversed-phase column packing, simply the polypeptide phase transition critical value) (There is also a phase transition of the adsorption ability of the polypeptide to the reverse phase column packing material). Specifically, when the volume ratio of the organic solvent in the mixed solution in which the polypeptide is dissolved exceeds the critical value of the phase transition of the adsorption ability to the reversed-phase column filler, the polypeptide Becomes a polypeptide that does not substantially exhibit the adsorption ability to the reverse phase column packing material (OFF phase polypeptide), and conversely, when the volume ratio is lower than the phase transition critical value of the adsorption ability to the reverse phase column packing material. Was found to be a polypeptide (ON-phase polypeptide) exhibiting adsorption ability to the reverse-phase column packing material. In addition, the present inventors also apply a reverse phase column packing material to a substance (excluding the reverse phase column packing material) that comes into contact with the polypeptide when the polypeptide sample is diluted and measured using a reverse phase liquid chromatograph. The polypeptide showed the same behavior as the case, and it was found that the phase transition critical value of the adsorptive capacity to these substances and the phase transition critical value of the adsorbing capacity to the column packing material are approximate (see Example 1).

  Furthermore, the present inventors have determined that the phase transition critical value of the adsorption ability of the polypeptide to the reversed-phase column packing material is the kind of the organic solvent contained in the solution in which the polypeptide is dissolved (see Example 2) and the polypeptide ( The phase of the adsorption phase of the polypeptide to the reversed-phase column packing material depends on the stationary phase of the reversed-phase column (see Example 8) and the temperature of the reversed-phase column (see Example 9). It has been found that the effect on the transition is small compared to the effect of the organic solvent.

  Further, the present inventors dissolve in a mixed solution Mx composed of water and one or more organic solvents (organic solvent 1, organic solvent 2,..., Organic solvent n (n is an integer of 1 or more)). It has been found that the adsorption ability of the polypeptide to the reversed-phase column packing conforms to the following formula (a) (see Examples 1 to 4).

(In the formula (a), X1 is a critical value of phase transition of polypeptide A in a water-organic solvent 1 mixed solution (%), Xn is a critical value of phase transition of polypeptide A in a mixed solution of water-organic solvent n (%)) X1 represents the volume ratio (%) of the organic solvent 1 in the mixed solution Mx, and xn represents the volume ratio (%) of the organic solvent n in the mixed solution Mx).

  Therefore, the present inventors utilize such knowledge, and in a method for quantifying a certain polypeptide (polypeptide A) using a reverse phase liquid chromatograph, the polypeptide A is substantially compared to the reverse phase column packing material. Polypeptide A (OFF-phase polypeptide A), which is a non-interacting phase (that is, a phase that does not substantially interact with a substance that is in contact with the preparation), is introduced into the reversed-phase liquid chromatograph and introduced. The phase of the OFF-phase polypeptide A is changed to a phase that interacts with the reverse-phase column packing, and then the ON-phase polypeptide A that is the phase that interacts with the column packing generated by the phase transition is added to the column. By interacting with the filler, a small amount of polypeptide at a low concentration where adsorption to a container or the like poses a problem can be easily and accurately determined without limitation on the amount of sample injection. Heading to be able to, it has led to the completion of the present invention.

That is, the present invention provides a method for detecting or quantifying a certain polypeptide (polypeptide A) using a reverse phase liquid chromatograph, which includes the following steps.
(1) introducing OFF-phase polypeptide A into a reversed-phase liquid chromatograph;
(2) a step of producing an ON phase polypeptide A by means of phase transition of the OFF phase polypeptide A introduced in (1),
(3) a step of allowing the ON phase polypeptide A produced in (2) to interact with the column filler,
(4) a step of producing an OFF-phase polypeptide A by causing phase transition of the ON-phase polypeptide A interacted in (3),
(5) a step of eluting the OFF phase polypeptide A produced in (4), and (6) a step of detecting or quantifying the polypeptide A eluted in (5).

  By carrying out the present invention, it is possible to quantitatively manipulate a trace amount of a polypeptide, for example, a polypeptide having a low pM level, and at the same time, it is possible to quantify easily and accurately. The present invention is a polypeptide quantification method capable of high-sensitivity quantification comparable to or higher than immunological methods widely used as conventional quantification methods for trace polypeptides, for example, quantification methods such as ELISA. It is an epoch-making invention that can be used in a wide range of fields such as medicine, biochemistry, and molecular biology.

It is a figure which shows the example of the reverse phase liquid chromatograph system for polypeptide quantification. The conventional system (A) and the present system (C) when using two pumps (mobile phase feeders), the conventional system (B) and the present system (D) when using three pumps. Urocortin peak area when measuring 1 nM urocortin sample solution with different acetonitrile content and added acid using _C30 column and conventional system. C ₄ column and a mass chromatogram obtained by measuring the different urocortin sample solution (including the volume ratio of 4% acetic acid) of acetonitrile content using conventional systems. Acetonitrile content in the sample solution: (A) 0%, (B) 20%, (C) 30%, (D) 40%, (E) 60%, and (F) 80%. It is a conceptual diagram of the elution behavior of the polypeptide sample solution introduced by the conventional system. It is a figure which shows the peak area of retention time 1.5 minutes and the peak area of retention time 6.5 minutes at the time of measuring the sample solution from which an acetonitrile content differs using a conventional system (black circle) or this invention system (white circle). . It is a figure which shows the power law of the gradient gradient and the retention time of urocortin. Peak shape near the baseline of urocortin measured using the conventional system (left) and the method of the present invention (right). Acetonitrile content in the sample solution: (A) 0%, (B) 20%, (C) 30%, (D) 40%, (E) 50%, (F) 60%, and (G) 80%. It is a figure which shows the mass chromatogram in the minimum determination of each polypeptide. It is a figure which shows the mass chromatogram in the minimum determination of each polypeptide. Total mass chromatogram (conventional system) of 18 kinds of polypeptide mixed solutions (1, 10 and 100 nM) having different solution compositions (conventional system) solution composition: (A) water, (B) acetic acid-water (4:96, v / v), (C) Acetic acid-water-acetonitrile (4:66:30, v / v / v), (D) Acetic acid-water-acetonitrile (4:46:50, v / v / v). Total mass chromatogram (system of the present invention) at the time of measurement of 18 kinds of polypeptide mixed solutions (1, 10 and 100 nM) having different solution compositions Solution composition: (A) Water, (B) Acetic acid-water (4:96, v / v), (C) acetic acid-water-acetonitrile (4:66:30, v / v / v), (D) acetic acid-water-acetonitrile (4:46:50, v / v / v). Isoleucil-seryl-bradykinin (top) and urocortin (bottom) of 18 polypeptide mixed solutions (1, 10 and 100 nM) having different solution compositions using the conventional system (left) and the developed system (right) It is a figure which shows a peak area. The figure which shows CD spectrum of urocortin in 20 degreeC (A) and 40 degreeC (B) water-acetonitrile mixed solution (volume ratio: 90:10, 80:20, 70:30, 60:40 or 50:50). It is. CD of isoleucyl-seryl-bradykinin in water-acetonitrile mixed solution (volume ratio: 100: 0, 95: 5, 90:10, 85:15 or 80:20) at 20 ° C (A) and 40 ° C (B) It is a figure which shows a spectrum.

  The present invention is a method for detecting or quantifying a polypeptide using a reverse phase liquid chromatograph.

  Here, reverse phase liquid chromatography includes all substances in which the mobile phase (eluent) is a liquid and the stationary phase is a substance that is less polar than the mobile phase (eluent). Examples of the stationary phase include hydrophobic groups such as hydrocarbon groups, for example, triacontyl group (C30), octadecyl group (C18) octyl group (C8), butyl group (C4), or trimethyl group. Examples of the support include inorganic porous polymers such as silica gel, polystyrene, methacryl, vinyl alcohol, hydroxyapatite, and titanium oxide. Graphite carbon can also be used as a filler. A person skilled in the art can appropriately select a filler according to the test polypeptide.

  A liquid chromatograph / mass spectrometer (LC-MS) is preferably exemplified as the liquid chromatograph. As the liquid chromatograph / mass spectrometer (LC-MS), a liquid chromatograph / tandem mass spectrometer (LC-MS / MS) is preferably exemplified. By using a reverse phase liquid chromatograph to which a mass spectrometer as a polypeptide detection or quantification apparatus is connected, it is possible to detect or quantify a highly sensitive polypeptide.

  The test polypeptide is not limited to molecular weight, isoelectric point, function, structure, etc., and includes all polypeptides. Among these, when high-sensitivity quantification is performed using LC-MS / MS, it is difficult to achieve high sensitivity when the number of multivalent ions generated in the ESI ion source is large, so that the molecular weight is about 10,000 Da or less. Certain polypeptides are preferred. The test polypeptide includes either a known polypeptide or an unknown polypeptide. Here, the polypeptide is a generic term including any of protein, polypeptide and oligopeptide, and the minimum size is 2 amino acids. The test polypeptide is not limited to biologically derived polypeptides such as various tissues, cells, bacteria and viruses, but is a polypeptide synthesized by a known synthesis method, a polypeptide obtained by a known genetic engineering technique, and various proteases. It includes both polypeptide fragments of certain proteins produced by enzymatic digestion and polypeptides that can be purchased as standard products. In addition, the test polypeptide may be prepared by a known method such as centrifugation, denaturation, fractionation using ammonium sulfate, purification treatment using dialysis, ultrafiltration, ion chromatography, or the like. It may be a polypeptide contained in a sample prepared by the method. Furthermore, in the case of obtaining a polypeptide from a biological sample, for example, a sample such as blood, urine, saliva, semen, spinal fluid, tissue, cell, etc., the sample can be subjected to a known pretreatment. The test polypeptide contained in the test polypeptide sample may be one type or two or more types. That is, using this method, it is possible to simultaneously detect or quantify multiple analyte polypeptides (see Example 13).

  For example, a polypeptide comprising the first to 24th amino acid sequences of adrenocorticotropic hormone, a polypeptide comprising the first to sixteenth amino acid sequences of β-amyloid, and the first to 28th amino acid sequences of β-amyloid A polypeptide consisting of the amino acid sequence of β-amyloid from the 1st to the 38th amino acid sequence, a polypeptide consisting of the amino acid sequence of the β-amyloid from the 1st to the 40th amino acid, the amino acid sequence of the β-amyloid from the 1st to the 42nd amino acid sequence Polypeptide consisting of the first amino acid, polypeptide consisting of the first to the 43rd amino acid sequence of β amyloid, growth hormone-releasing factor, isoleucyl-serirubradykinin, brain natriuretic peptide (BNP-32), insulin, C-type sodium Diuretic peptide (CNP-53), Mid Polypeptide consisting of amino acids 60 to 121 of in, neuromedin C, neuropeptide Y, nociceptin, oxytocin, urocortin, midkine, interferon-γ, atrial natriuretic peptide (ANP (1-28)), Rat neutrophil chemotactic factor-1 (CINC-1 / gro), parathyroid hormone (PTH (1-84)), a polypeptide consisting of amino acids 323 to 339 of ovalbumin, ovalbumin, angiotensin II Examples include angiotensin II in which the fourth tyrosine of the amino acid sequence is phosphorylated (see Examples 5 to 6).

  In the present invention, the OFF phase polypeptide means a polypeptide in an OFF phase state. The OFF phase state means that the polypeptide is present in the solution in a state in which the adsorption ability to the packing material of the column of the reverse phase liquid chromatograph is substantially lost. The state in which the adsorptive capacity is substantially lost means a state in which it is not adsorbed at all or a state in which it is hardly adsorbed as compared with a polypeptide in an ON phase state.

  The inventors of the present invention are not limited to a solid phase (hereinafter referred to as a container used during sample preparation). It has been found that the same behavior is exhibited. Furthermore, it was suggested that the adsorption ability of the polypeptide to the container or the like used during sample preparation was substantially equal to or slightly lower than the adsorption ability to the silica-based column packing (Example 1). Reference), it is considered that the OFF-phase polypeptide substantially loses the adsorption ability even for a container or the like used for sample preparation. Examples of containers and the like used during sample preparation include chips, sample vials, sample injectors, syringes, and liquid feeders. Examples of these materials include polypropylene, polytetrafluoroethylene, silicone, stainless steel, glass, PEEK resin, ceramic, Vespel, and Tefzel.

  The present inventors adsorbed 27 kinds of test polypeptides to the column packing material in each of the mixed solutions of water-acetonitrile mixed solution, water-ethanol mixed solution, water-methanol mixed solution, and water-acetic acid mixed solution. An approximation of the phase transition critical value of Noh was determined (see Example 5). From this result, a water-acetonitrile mixed solution in which the volume ratio of acetonitrile is 50% or more, a water-ethanol mixed solution in which the volume ratio of ethanol is 60% or more, and a water-methanol mixture in which the volume ratio of methanol is 70% or more In each mixed solution of the solution and the water-acetic acid mixed solution in which the volume ratio of acetic acid is 70% or more, it was revealed that all 27 kinds of test polypeptides were in the OFF phase. Similarly, other polypeptides are considered to be in the OFF phase in these mixed solutions. Therefore, the volume ratio of acetonitrile is 50% or more, the volume ratio of ethanol is 60% or more, the volume ratio of methanol is 70% or more and / or the volume ratio of acetic acid is 70% or more, and acetonitrile By dissolving the test polypeptide in a solution containing one or more organic solvents selected from methanol, ethanol and acetic acid, for example, a mixed solution of water-acetonitrile (volume ratio of water to acetonitrile is 1: 1). An OFF phase polypeptide sample can be prepared.

  Moreover, an OFF phase polypeptide sample can also be prepared according to the method as described in an Example, for example. Specifically, (1) 1 type, or 2 or more types of organic solvents are selected from the organic solvents which can be used for a reverse phase liquid chromatograph. Next, (2) for each selected organic solvent, the phase transition critical value of the test polypeptide in the mixed solution of water and the organic solvent (or a value exceeding the phase transition critical value) is determined. When the selected organic solvent is one kind, (3) the test polypeptide is added to a mixed solution of water and the organic solvent in which the volume ratio of the organic solvent sufficiently exceeds the determined phase transition critical value. By dissolving, an OFF phase polypeptide sample can be prepared. When two or more kinds of organic solvents are selected, (3) by dissolving the test polypeptide in a mixed solution showing the volume ratio of each organic solvent calculated according to the idea of the formula (a) (f> 1) An OFF phase polypeptide sample can be prepared.

  Examples of the organic solvent that can be used for the reverse phase liquid chromatograph include acetonitrile, methanol, ethanol, isopropyl alcohol, acetone, DMSO, THF, acetic acid, formic acid, TFA, and the like. Among these, acetonitrile, methanol, ethanol, isopropyl alcohol, acetic acid, formic acid, and TFA are preferable. These are used alone or in combination of two or more. The type of solvent used as the mobile phase and the type of solvent of the test polypeptide sample may be the same or different. For example, a water-acetonitrile mixed solvent may be used as the mobile phase, and the solvent of the test polypeptide sample may be a water-methanol and acetonitrile mixed solvent containing about several percent of acetic acid. In general, for a certain polypeptide, the phase transition critical value of the polypeptide decreases in the order of isopropyl alcohol> acetonitrile and ethanol> methanol and acetic acid (see Example 15). .

  The method for determining the phase transition critical value of the test polypeptide will be specifically described in the case where acetonitrile is selected as the organic solvent. A plurality of water-acetonitrile mixed solutions having different volume ratios of water and acetonitrile (for example, the volume ratio of water: acetonitrile is 10: 0, 9: 1, 8: 2, 7: 3, 6: 4, 5: 5 4: 6, 3: 7, 2: 8, 1: 9, 0:10) are prepared, and a plurality of test polypeptide samples having different volume ratios of water and acetonitrile are prepared. To do. Next, each test polypeptide sample is introduced into a reverse-phase liquid chromatograph (the conventional reverse-phase liquid chromatograph configuration described in FIG. 1 (A) or (B) can be exemplified), and each test polypeptide It is determined whether or not the test polypeptide contained in the sample is in the OFF phase. The mobile phase for introduction is preferably an aqueous mobile phase, and the mobile phase for elution is preferably an organic solvent mobile phase. The aqueous mobile phase is a mobile phase containing no organic solvent or slightly containing an organic solvent. Examples of the aqueous mobile phase include an organic acid aqueous solution of about 0.01 to 6%. The organic solvent-based mobile phase means a mobile phase consisting of only one or more kinds of organic solvents or a mobile phase consisting of one or more kinds of organic solvents containing slightly water. As an organic solvent system mobile phase, for example, an acetonitrile-methanol mixed solution containing about 0.01 to 6% organic acid, an acetonitrile solution containing about 0.01 to 6% organic acid, about 0.01 to 6% Examples include methanol solutions containing organic acids, ethanol solutions containing about 0.01 to 6% organic acids, and the like. Preferred examples of the organic acid include acetic acid, formic acid, and TFA. Moreover, a 100% acetic acid solution can be illustrated as an organic solvent system mobile phase. When the test polypeptide is in the OFF phase, the test polypeptide (or a part thereof) does not adsorb to the column packing material, so that an elution peak is detected at the retention time corresponding to the dead volume after the sample loop (see Example 1). ). For example, the polypeptide contained in each test polypeptide sample in which the volume ratio of water: acetonitrile is 6: 4, 5: 5, 4: 6, 3: 7, 2: 8, 1: 9, 0:10 is OFF. In the case of the phase, the phase transition critical value of the test polypeptide in the water-acetonitrile mixed solution can be determined to be 30 to 40%. In this case, an OFF-phase polypeptide sample can be prepared by dissolving the polypeptide in a water-acetonitrile mixed solution containing 40% or more acetonitrile.

  A method for preparing an OFF phase polypeptide sample will be specifically described in the case where acetonitrile and methanol are selected as the organic solvent. According to the above-mentioned method, the phase transition critical value of the test polypeptide in each of the water-acetonitrile mixed solution and the water-methanol mixed solution is determined. Next, the volume ratio of acetonitrile and methanol in the sample is determined according to the idea of the formula (a). For example, when the phase transition critical value of the test polypeptide in the water-acetonitrile mixed solution is determined to be 30 to 40% and the phase transition critical value of the test polypeptide in the water-methanol mixed solution is determined to be 40 to 50%, water: acetonitrile Since the value of the above formula (a) for the test polypeptide in a water-acetonitrile and methanol mixed solution having a volume ratio of methanol: 1: 1: 8 exceeds 1, the test polypeptide is dissolved in the mixed solution Thus, an OFF phase polypeptide sample can be prepared.

  When analyzing a test polypeptide sample by a conventional reverse phase liquid chromatograph (FIG. 1 (A)) using two types of mobile phases (an aqueous mobile phase and an organic solvent mobile phase), the present inventors It was found that a power law is established between the retention time of the peptide and the gradient gradient (see Example 6). Furthermore, the following formula (b) is established between the elution time by two types of gradient gradients, their gradient gradients, and the mobile phase (eluent) composition at the time of elution of the test polypeptide by each gradient gradient. (See Example 6). However, at this time, the aqueous mobile phase is a mobile phase that hardly contains an organic solvent and an organic acid, the organic solvent-based mobile phase is a mobile phase that does not contain water, and mixing at the start of measurement of these mobile phases. This is the case when the ratio is measured under conditions where the aqueous mobile phase: organic mobile phase = 100: 0.

(In the formula (b), r ₁ is a gradient gradient (% / min), r ₂ is a gradient gradient (% / min) different from r _1, and T ₁ is the elution time of the test polypeptide by the gradient gradient r ₁ ( min), T ₂ is the elution time (min) of the test polypeptide by the gradient gradient r ₂ , and V is the volume ratio (%) of the organic solvent mobile phase in the mobile phase (eluent) at the time of elution of the test polypeptide. Show.)

  The eluted test polypeptide is considered not to have the ability to adsorb to the column packing material in the mobile phase (eluent) at the time of elution. Therefore, it is also possible to prepare an OFF-phase polypeptide sample by dissolving the test polypeptide in a mixed solution in which the volume ratio of the aqueous mobile phase and the organic solvent-based mobile phase is (100-V): V.

When analyzing a test polypeptide sample by a conventional reverse phase liquid chromatograph (FIG. 1 (A)) using two types of mobile phases (an aqueous mobile phase and an organic solvent mobile phase), the present inventors It was found that a power law is established between the retention time of the peptide and the gradient gradient (see Example 6). When the power function is approximated by y = Bx− ^t (y: retention time, x: gradient gradient, t: power exponent, where t> 0), the constant B is the retention time (elution at a gradient gradient of 1% / min) Time). In addition, the elution time of the test polypeptide at a gradient gradient of 1% / min is the rate at which the mobile phase for elution of the test polypeptide increased in the mobile phase (in the eluent) from the time of introduction of the test polypeptide to the time of elution. (%) (However, since the increase includes dead volume, it is an approximate value). Using this property, for example, an aqueous solution containing no organic solvent is used as the aqueous mobile phase, and one kind of organic solvent selected as the organic solvent mobile phase is used. : Organic mobile phase = When measured under the condition of 100: 0, the elution time of the test polypeptide with a gradient gradient of 1% / min is the phase transition criticality of the test polypeptide in the water-organic solvent mixture solution. It is also possible to approximate the value (%) (see Example 7). Using this knowledge, an OFF-phase polypeptide sample can be easily prepared.

  In the present invention, as described above, first, (1) a test polypeptide (polypeptide A) is introduced into a reversed-phase liquid chromatograph in an OFF phase state, and (2) then, means for causing phase transition of the OFF phase polypeptide A Is performed to cause the phase transition of the OFF phase polypeptide A to the ON phase polypeptide A, and (3) the ON phase polypeptide A is retained in the column by interacting with the column packing material. (4) The ON phase polypeptide A retained on the column is phase-transduced to convert the ON phase polypeptide to the OFF phase polypeptide A, and (5) the OFF phase polypeptide A is eluted. ) Detect or quantify the eluted polypeptide A. Hereinafter, the steps (1) to (6) will be described.

  Step (1) is a step of introducing the OFF phase polypeptide A into the reverse phase liquid chromatograph. In the conventional reverse phase liquid chromatography, it has not been studied at all whether the test polypeptide is in the OFF phase state or the ON phase state. For example, as in Example 1 described later, urocortin has a very weak adsorption ability to the column packing in a solution having a high acetonitrile content (40% or more), but rapidly when the acetonitrile content is less than 30%. It has been found for the first time by the inventor's investigation that the adsorption capacity to the column packing is strong (see Example 1). From this, it is considered that the phase transition critical value of the adsorption ability of urocortin to the column filler in the water-acetonitrile solution is between 30% and 40%. Further, it was suggested that the adsorption ability of the polypeptide to the container or the like used during sample preparation is substantially equal to or slightly lower than the adsorption ability to the silica-based column filler. For example, as in Example 1 described later, for urocortin, the peak area was almost constant when the acetonitrile content in the sample solution was 30% or more, so the acetonitrile content in the sample solution was 30% or more. In this case, it was considered that the adsorption to the container or the like used during sample preparation hardly occurred from immediately after the sample preparation to the start of measurement. However, even when the acetonitrile content is 30%, there is a possibility that adsorption to the container or the like may occur when left in the container or the like for a long time. Therefore, a polypeptide sample having an organic solvent content lower than the phase transition critical value of the adsorptive capacity is partially lost due to adsorption to a container or the like and loses its quantitative property. Therefore, in step (1), the test polypeptide In order to introduce into a reversed-phase liquid chromatograph without loss before introduction, it is considered that adsorption to a container or the like can be avoided if the test polypeptide contained in the test polypeptide sample is in the OFF phase.

  The OFF phase polypeptide sample can be prepared according to the preparation method of the sample described above. When a polypeptide is detected or quantified by this method, the polypeptide preferably has a phase transition critical value of about 5 to 95%, and the polypeptide has a phase transition critical value of 10 to 90%. More preferably, it is about. When the critical value of phase transition of the polypeptide is not within this range, it is preferable to change the type of organic solvent in the sample or mobile phase, column filler, column temperature, and the like. Such changes can be appropriately made by those skilled in the art.

  The sample can be introduced from a sample injector into a reverse phase liquid chromatograph. The mobile phase for introducing this sample is not particularly limited, but an organic solvent-based mobile phase is preferable. The organic solvent-based mobile phase is preferably such that when the test polypeptide is dissolved in the mobile phase, the test polypeptide becomes an OFF phase. As an organic solvent system mobile phase, for example, an acetonitrile-methanol mixed solution containing about 0.01 to 6% organic acid, an acetonitrile solution containing about 0.01 to 6% organic acid, about 0.01 to 6% Examples include methanol solutions containing organic acids, ethanol solutions containing about 0.01 to 6% organic acids, and the like. Acetic acid can be preferably exemplified as the organic acid. Moreover, a 100% acetic acid solution can be illustrated as an organic solvent system mobile phase.

  Step (2) is a step of causing the phase transition of the OFF phase polypeptide A to the ON phase polypeptide A by executing a means for phase transition of the OFF phase polypeptide A. The phase transition produces ON phase polypeptide A. The produced ON phase polypeptide A is adsorbed on the column packing material.

  The ON-phase polypeptide may be adsorbed not only to the column filler but also to a container or the like used during sample preparation. Therefore, the step (2) is preferably a stationary phase, that is, immediately before the column (FIGS. 1C and 1D).

  In the present invention, the means for causing the phase transition of the OFF phase polypeptide A means a means for producing or promoting the production of the ON phase polypeptide from the OFF phase polypeptide A contained in the mobile phase.

  The means for causing the phase transition of the OFF phase polypeptide A will be specifically described. The phase transition of the OFF phase polypeptide A is a mobile phase in which the OFF phase polypeptide A exists (an organic solvent contained in the mobile phase is an organic solvent 1, 2,..., An organic solvent n (n is 1 In the above formula (a), the f value in the above-described formula (a) is a means for making the value smaller than 1. By changing the type of solvent and / or the composition of the solvent contained in the mobile phase in which the OFF-phase polypeptide A is present, the f value can be made smaller than 1. By adding and stirring a mobile phase different from the mobile phase to the mobile phase in which the OFF-phase polypeptide A is present (sometimes referred to as a mobile phase for polypeptide A phase transition), the OFF-phase polypeptide A The type of solvent and / or the composition of the solvent contained in the mobile phase containing can be changed. The mobile phase in which the OFF-phase polypeptide A before this change exists may be referred to as a polypeptide A-introducing mobile phase. The mobile phase for polypeptide A phase transition is a mobile phase (ON phase solution) in which polypeptide A becomes an ON phase when polypeptide A is dissolved in the mobile phase.

  By carrying out a means for causing the phase transition of the OFF-phase polypeptide A, the f value can be made smaller than 1. By making the f value smaller than 1, the OFF phase polypeptide A contained in the mobile phase undergoes a phase transition to produce the ON phase polypeptide A.

  The means for phase transition of the OFF phase polypeptide A by adding and stirring the polypeptide A phase transition mobile phase to the polypeptide A introduction mobile phase will be described more specifically. However, the OFF-phase polypeptide A sample is a sample in which polypeptide A is dissolved in a mixed solvent composed of water, organic solvent X, organic solvent Y, and organic solvent Z, and the mobile phase for introducing polypeptide A is water, organic solvent L A case where the mobile phase is composed of organic solvent M and organic solvent N, and the mobile phase for polypeptide A phase transition is a mobile phase composed of water, organic solvent O, organic solvent P, and organic solvent Q will be described.

  The organic solvent used in the mobile phase is appropriately selected from organic solvents that can be used in the reverse phase liquid chromatograph to be used, such as acetonitrile, methanol, ethanol, isopropyl alcohol, acetone, DMSO, THF, acetic acid, formic acid, and TFA. Can do. These can be used alone or in combination of two or more.

  The addition and stirring of the mobile phase for polypeptide A phase transition can be carried out, for example, with a mixer (FIGS. 1C and 1D) provided in a reverse phase liquid chromatograph.

  Specific method for determining the mixing ratio of the mobile phase for introducing polypeptide A and the mobile phase for transferring phase of polypeptide A (mobile phase for introducing polypeptide A: mobile phase for transferring polypeptide A = 1: α) Explained.

  In this example, water-organic solvent X mixed solution, water-organic solvent Y mixed solution, water-organic solvent Z mixed solution, water-organic solvent L mixed solution, water-organic solvent M mixed solution, water-organic solvent N mixed The phase transition critical values of polypeptide A in each of the solution, the water-organic solvent O mixed solution, the water-organic solvent P mixed solution, and the water-organic solvent Q mixed solution are X (%) and Y (% ), Z (%), L (%), M (%), N (%), O (%), P (%), and Q (%). These phase transition critical values can be determined by the aforementioned method for determining the phase transition critical value. In this example, the volume ratios of water, organic solvent X, organic solvent Y, and organic solvent Z in the OFF phase polypeptide A sample are w1 (%), x (%), y (%), and z (%), respectively. (W1 + x + y + z = 100). In this example, the volume ratios of water, organic solvent L, organic solvent M, and organic solvent N in the mobile phase for introducing polypeptide A are w2 (%), l (%), m (%), and n (%), respectively. ) (Where w2 + l + m + n = 100). In this example, the volume ratios of water, organic solvent O, organic solvent P, and organic solvent Q in the mobile phase for polypeptide A phase transition are w3 (%), o (%), p (%), q ( %) (Where w3 + o + p + q = 100).

  Since polypeptide A present in the OFF-phase polypeptide A sample is in the OFF phase, x / X + y / Y + z / Z> 1 is established according to the above-described formula (a).

  When the polypeptide A is dissolved in the mobile phase for phase transition of the polypeptide A, the polypeptide A becomes an ON phase. Therefore, according to the idea of the above formula (a), o / O + p / P + q / Q <1 Is established. Those skilled in the art can easily prepare such a mobile phase for polypeptide A phase transition. For example, when O = 20%, P = 40%, and Q = 50%, all of o, p, and q may be 5%.

  When polypeptide A present in the mobile phase for introducing polypeptide A is in the OFF phase (that is, when the mobile phase for introducing polypeptide A is the OFF phase solution), 1 / L + m / M + n / N> 1 is established. Yes. Those skilled in the art can easily prepare such a mobile phase for introducing polypeptide A. For example, when O = 20%, P = 40%, and Q = 50%, o, p, and q may all be set to 20%. The polypeptide A sample introduced into the reversed-phase liquid chromatograph is transferred for subsequent polypeptide A introduction in the pipe while diffusing before and after, unless it is stirred and mixed with the mobile phase for polypeptide A introduction by a mixer or the like. It is considered that the mobile phase in the vicinity of polypeptide A almost holds the solvent composition of the introduced polypeptide A sample (FIGS. 4B and 4C). Accordingly, in a solution obtained by mixing the polypeptide A sample and the polypeptide A phase transition mobile phase at 1: α, the polypeptide A becomes the ON phase, and the polypeptide A introduction mobile phase and the polypeptide A phase transition It is considered that polypeptide A needs to be in the ON phase even in a solution in which the mobile phase is mixed at 1: α. From this idea, β = {(l / L + m / M + n / N) −1} / {1- (o / O + p / P + q / Q)} and γ = {(x / X + y / Y + z / Z) −1}. Assuming / {1- (o / O + p / P + q / Q)}, α is a larger value than β and γ (β or γ when β and γ are the same) or more.

  When the polypeptide A present in the mobile phase for introducing polypeptide A is the ON phase (that is, when the mobile phase for introducing polypeptide A is the ON phase solution), 1 / L + m / M + n / N <1 is established. Yes. Those skilled in the art can easily prepare such a mobile phase for introducing polypeptide A. For example, when O = 20%, P = 40%, and Q = 50%, o, p, and q may all be set to 5%. In this case, it is considered that the polypeptide A needs to be in the ON phase in a solution obtained by mixing the polypeptide A sample and the mobile phase for polypeptide A phase transition at 1: α. From this idea, α is the above-mentioned γ.

  The mixing ratio will be further described with urocortin as an example of the test polypeptide. In Example 1 described later, the phase transition critical value of urocortin in the water-acetonitrile mixed solution is about 35%, the phase transition critical value of urocortin in the water-ethanol mixed solution is about 45%, and urocortin in the water-acetic acid mixed solution is It was revealed that the phase transition critical value was about 65% (see Example 3). Therefore, the solvent of the OFF phase urocortin sample is a solvent composed of water, acetonitrile and ethanol, and a volume ratio of water: acetonitrile: ethanol is 20%: 35%: 45%. As a mobile phase for introducing urocortin, a solvent comprising water, acetic acid, acetonitrile and ethanol, wherein the volume ratio of water: acetic acid: acetonitrile: ethanol is 16.75%: 3.25%: 35%: 45% Can be illustrated. An example of a mobile phase for urocortin phase transition is a 3.25% aqueous acetic acid solution. In this example, β = {(3.25 / 65 + 35/35 + 45/45) -1} / {1- (3.25 / 65)}, γ = {(35/35 + 45/45) -1} / {1 − (3.25 / 65)}. That is, β is about 1.1 and γ is about 1.05. Therefore, α can be calculated to be about 1.1 or more. For example, the mobile phase for introducing urocortin and the mobile phase for urocortin phase transition may be mixed and stirred at a mixing ratio of 1: 2.

  That is, as a means for causing the phase transition of the OFF phase polypeptide A, a means for reducing the organic solvent content in the mobile phase containing the OFF phase polypeptide can be exemplified. By increasing the water content of the mobile phase containing the OFF phase polypeptide, the organic solvent content in the mobile phase containing the OFF phase polypeptide can be reduced.

The present inventors used 27 kinds of test polypeptides as a target, using a water-isopropyl alcohol mixed solution, a water-acetonitrile mixed solution, a water-ethanol mixed solution, a water-methanol mixed solution, and a water-acetic acid mixed solution, As a result of calculating the approximate value of the phase transition critical value in each organic solvent (including acetic acid), it was found to be about 5 to 70% (see Example 15). Regarding the polypeptides other than the 27 types of polypeptides used this time, it is considered that there are cases where the phase transition critical value is about 1 to 5%. Therefore, by reducing the content of each organic solvent contained in the eluent to a critical value or less (f <1), for example, about 0.01 to 1% or less, preferably about 0.01 to 0.5% or less. It is considered that an OFF phase polypeptide can be phase-shifted to produce an ON phase polypeptide.
On the other hand, when the column pressure was high, it was found that there is a case where a polypeptide having a short retention time elutes early from the column (Examples 7 and 15). This is presumably because the higher-order structure of the polypeptide changes to a higher-order structure having a smaller critical value due to a high column pressure.

  Mobile phase supply (mobile phase 1 supply) that supplies at least a mobile phase (mobile phase 1), mobile phase supply (mobile phase 2 supply) that supplies a mobile phase (mobile phase 2) different from mobile phase 1 ), A sample injector connected to the mobile phase 1 supply device via a liquid feeding tube, a mobile phase mixer connecting the mobile phase 2 supply device and the sample injector via a liquid feeding tube, and the mobile phase When using a reversed-phase liquid chromatograph (FIG. 1 (C)) having a reversed-phase analysis column connected to a mixer via a liquid feeding tube and a polypeptide detection or quantification device connected to the reversed-phase analysis column The step (2) will be described.

Step (2) is performed by mixing and stirring the mobile phase 1 and the mobile phase 2 using a mobile phase mixer. The mobile phase 1 may be a mobile phase in which when the test polypeptide is dissolved in the mobile phase 1, the test polypeptide becomes an OFF phase. As such a mobile phase, isopropyl alcohol is 40% or more, acetonitrile volume ratio is 50% or more, ethanol volume ratio is 50% or more, methanol volume ratio is 80% or more, and / or acetic acid volume ratio is 80%. A solution containing one or more organic solvents selected from isopropyl alcohol, acetonitrile, methanol, ethanol and acetic acid, for example, water-acetonitrile (volume ratio of water to acetonitrile is 1: 1) A mixed solution can be illustrated. Examples of the mobile phase 2 include water or an aqueous organic acid solution of several percent. Preferred examples of the organic acid include acetic acid, formic acid, and TFA. Step (2) can be carried out by mixing and stirring the mobile phase 1 and the mobile phase 2 with a mobile phase mixer at a mixing ratio of 1: 100 to 1: 1, preferably 1: 100 to 1: 2.
More specifically, for example, in a mobile phase mixer, mobile phase 1 which is an acetonitrile-methanol mixture (volume ratio 1: 1) containing acetic acid having a volume ratio of 4%, and an acetic acid aqueous solution having a volume ratio of 4%. Step (2) can be performed by mixing and stirring the mobile phase 2 at a mixing ratio of 2: 8 (see Example 1).

  Although the stationary phase of the column and the column temperature also affect the retention time of the polypeptide in the stationary phase (see Examples 8 and 9), there is a phase transition of the adsorption capacity of the polypeptide in the polypeptide solution to the column packing material. Compared to the effect, it is slight. In general, increasing the column temperature shortens the retention time of the polypeptide in the stationary phase, so the transition critical value of the polypeptide decreases. Conversely, decreasing the column temperature decreases the polypeptide stationary phase. Since the retention time in the polypeptide becomes longer, the critical value of phase transition of the polypeptide increases. In general, since the graphite carbon filler has a higher polypeptide retention than the silica gel filler, for a certain polypeptide, the phase transition critical value of the polypeptide when the silica gel filler is used is This is lower than the critical value of phase transition of the polypeptide when a graphite carbon filler is used.

  Step (3) is a step of allowing the ON phase polypeptide A to interact with the column filler. By the step (2), the test polypeptide is in a state capable of being adsorbed to the ON-phase polypeptide A, that is, the column packing material, and therefore is surely adsorbed to the entire amount packing material. Conventionally, as described above, it is not known that the adsorption ability of the polypeptide is phase-shifted between the OFF phase and the ON phase, so that the polypeptide introduced in the OFF phase state does not adsorb to the column packing material. In some cases, two peaks were generated (see Example 1). On the other hand, the polypeptide introduced in the ON phase is surely adsorbed to the column packing material, but part of it is lost by adsorption to the container before introduction, resulting in loss of quantitativeness and high sensitivity quantification. It was difficult.

  Step (4) is a step of producing an OFF phase polypeptide A by causing phase transition of the ON phase polypeptide A adsorbed on the filler. This step is performed by changing the concentration of organic solvent-water in the mobile phase, that is, by applying a gradient. What is necessary is just to raise the organic-solvent content in a mobile phase until this mobile phase turns into an OFF phase according to the said consideration. Compared to retention of low molecular weight compounds in column packing, retention of polypeptides in column packing is most affected by adsorption capacity in the presence of various interactions such as hydrophobic interactions. Is a feature. The adsorption ability of this polypeptide is characterized by a significant change at the boundary of the phase transition critical value, and the ON phase polypeptide adsorbed on the column packing is a low molecular weight that interacts with the column packing. Compared to compounds, it has the property of being difficult to move in the column under isocratic conditions. Using this property, the steps (1) to (3) can be repeatedly executed, and then the step (4) can be executed. As a result, the polypeptide can be adsorbed to the column filler up to the load limit of the polypeptide of the column filler, and highly sensitive quantification is possible.

  Step (5) is a step of eluting off-phase polypeptide A. Elution may be performed using an eluent that increases the concentration of the organic solvent in the eluent, that is, a solution composition in which the polypeptide becomes an OFF-phase polypeptide.

  Step (6) is a step of detecting or quantifying the eluted polypeptide A, and the detection may be any of UV, fluorescence, photodiode array and the like. For high-sensitivity determination, mass spectrum, particularly tandem mass spectrometry is preferred.

  Further, the present invention includes at least a mobile phase supply device (mobile phase 1 supply device) that supplies a certain mobile phase (mobile phase 1) and a mobile phase that supplies a mobile phase (mobile phase 2) different from mobile phase 1. A feeder (mobile phase 2 feeder), a sample injector connected to the mobile phase 1 feeder via a liquid feeding tube, and a movement connecting the mobile phase 2 feeder and the sample injector via a liquid feeding tube There is also provided a reverse phase liquid chromatograph having a phase mixer, a reverse phase analysis column connected to the mobile phase mixer via a liquid feeding tube, and a polypeptide detection or quantification device connected to the reverse phase analysis column. (FIG. 1C). This reverse phase liquid chromatograph can be used in the present detection or quantification method. The reverse-phase analysis column and the polypeptide detection or quantification device can be connected via a liquid feeder. A polypeptide detection or quantification device is a detection or quantification device capable of detecting or quantifying a polypeptide.

  A liquid chromatograph / mass spectrometer (LC-MS) is preferably exemplified as the reverse phase liquid chromatograph. As the liquid chromatograph / mass spectrometer (LC-MS), a liquid chromatograph / tandem mass spectrometer (LC-MS / MS) is preferably exemplified. By using a reverse phase liquid chromatograph to which a mass spectrometer as a polypeptide detection or quantification apparatus is connected, it is possible to detect or quantify a highly sensitive polypeptide. Therefore, a mass spectrometer, preferably a tandem mass spectrometer, can be used as a polypeptide detection or quantification device.

  In the case of using a mixture of two or more organic solvents and water as the mobile phase, a reverse phase liquid chromatograph having three or more mobile phase supply devices is preferable (FIG. 1D). Accordingly, at least a mobile phase supply device (mobile phase 1 supply device) that supplies a mobile phase (mobile phase 1) and a mobile phase supply device (mobile phase 2) that supplies a mobile phase (mobile phase 2) different from mobile phase 1 2), a mobile phase supplier (mobile phase 3 supplier) for supplying a mobile phase (mobile phase 3) different from mobile phases 1 and 2, and a mobile phase 1 supplier and a mobile phase 2 supplier. A mobile phase mixer (mixer A) connected via a liquid tube, a sample injector connected to the mixer A via a liquid supply tube, a mobile phase 3 supply device and the sample injector are connected to a liquid supply tube. A mobile phase mixer (mixer B) to be connected via a reverse phase analysis column connected to the mixer B via a liquid feed pipe, and a mass spectrometer connected to the reverse phase analysis column via a liquid feed pipe Liquid chromatograph / mass spectrometer (LC-MS) or liquid chromatograph / tandem mass spectrometer (LC-) S / MS) are also included in the present invention.

  Further, the mass spectrometer may be connected to a switching valve instead of connecting the mass spectrometer and the analytical column via the liquid feeding tank, and the switching valve may be connected to the reverse phase analytical column via the liquid feeding tank. . The switching valve can determine whether or not the eluate from the analysis column is transferred to the mass spectrometer. For example, after the detection or quantification of a polypeptide is completed, when washing impurities adsorbed on the analytical column, the switching valve is operated to stop the eluate from the analytical column from moving to the mass spectrometer. Can do.

  FIG. 1 shows a comparison between the system of the present invention (FIGS. 1C and 1D) and the conventional system (FIGS. 1A and 1B).

  According to the method of the present invention, a polypeptide sample having a solution composition capable of avoiding adsorption to a container or the like can be quantified without loss. Therefore, it is possible to measure up to the detection limit of the apparatus, and it is possible to accurately quantify a polypeptide of fM order or less depending on the amount of sample introduced into the system.

  As described in Examples (especially Examples 17 to 22) described later, the present inventor can improve the solubility of a certain polypeptide in a sample by adding acetic acid to a biological sample containing the polypeptide. I found out that I can.

Examples of the biological sample include plasma, urine, each tissue homogenate and the like, and a plasma-derived sample is preferable.
Although it is not clear why the addition of acetic acid improves the solubility of polypeptides in biological samples, it inhibits the interaction between polypeptides in plasma and certain polypeptides, and polypeptides in plasma and certain polypeptides This is thought to be due to the inhibition of the aggregation with the. That is, by adding acetic acid, the interaction between the same or different polypeptides can be inhibited in vitro, and their aggregation can be inhibited.

  The action of improving the solubility of a polypeptide by the addition of acetic acid is particularly effective when an organic solvent is added to a biological sample containing the polypeptide. The organic solvent is preferably one or more selected from acetonitrile, methanol, ethanol and isopropyl alcohol. That is, the solubility of a certain polypeptide (polypeptide A) in the sample can be improved by adding an organic solvent and acetic acid to the plasma-derived sample. The sample having improved solubility in this manner is OFF phase polypeptide A.

The polypeptide may have a molecular weight of 10,000 Da or more. In particular, it is useful for improving the solubility of β-amyloid or a partial polypeptide thereof in a biological sample. Examples of the β amyloid partial polypeptide include the following (1) to (4).
(1) a polypeptide comprising the first to the 38th amino acid sequence of β-amyloid,
(2) a polypeptide comprising the first to the 40th amino acid sequence of β-amyloid,
(3) A polypeptide consisting of amino acid sequence 1st to 42nd amino acid sequence of β-amyloid, and (4) a polypeptide consisting of amino acid sequence 1st to 43rd amino acid sequence of β-amyloid.
The amount of acetic acid to be added is preferably 50% or more. When used in combination with an organic solvent, the amount of organic solvent is preferably 10% or more, and the amount of acetic acid is preferably 50% or more.

  EXAMPLES Next, although an Example is given and this invention is demonstrated still in detail, this invention is not limited to these Examples at all.

  The reagents, polypeptides, etc. used in the examples are shown below.

Reagents Acetonitrile for HPLC (Kanto Chemical)
Methanol for HPLC (Kanto Chemical)
Ethanol for HPLC (Kanto Chemical)
Isopropyl alcohol (2-propanol) for HPLC (Kanto Chemical)
Acetic acid for column chromatogram (Nacalai Tesque)
Formic acid for column chromatogram (Nacalai Tesque)
Trifluoroacetic acid (TFA; Nacalai Tesque)
Dimethyl sulfoxide (DMSO; Nacalai Tesque)
Ultrapure water (low total organic carbon water: Low TOC water)

Polypeptide (27 types)
The following polypeptides were purchased from Peptide Institute and used.
A polypeptide consisting of the first to 24th amino acid sequences of corticotropin (ACTH (1-24)) *
Polypeptide consisting of amino acid sequence 1 to 16 of β-amyloid (amyloid β-protein (1-16)) *
Polypeptide consisting of amino acid sequence 1 to 40 of β-amyloid (amyloid β-protein (1-40)) *
・ Polypeptide consisting of amino acid sequence 1st to 42nd amino acid sequence of β-amyloid *
(Amyloid β-protein (1-42)) *
Polypeptide consisting of amino acid sequence 1 to 43 of β-amyloid (amyloid β-protein (1-43)) *
・ Growth hormone releasing factor (GRF) *
* Isoleucyl-seryl-bradykinin *
-Brain natriuretic peptide (BNP-32) *
・ Insulin *
・ C-type natriuretic peptide (CNP-53) *
A polypeptide consisting of amino acids 60 to 121 of midkine (midkine (60-121)) *
・ Neuromedin C *
・ Neuropeptide Y (NPY) *
・ Nociceptin *
・ Oxytocin *
・ Urocortin *
Atrial natriuretic peptide (ANP (1-28))
・ Midkine
Rat neutrophil chemotactic factor-1 (CINC-1 / gro)
・ Parathyroid hormone (PTH (1-84))
The following polypeptides were obtained from the American Peptide Company, Inc. We purchased more and used.
Polypeptide consisting of amino acid sequence 1st to 28th amino acid sequence of β amyloid (amyloid β-protein (1-28)) *
Polypeptide consisting of amino acid sequence 1 to 38 of β-amyloid (amyloid β-protein (1-38)) *
(Polypeptides marked with * are referred to as 18 kinds of polypeptides in the examples described later)
The following polypeptides were purchased from Sigma and used.
・ Interferon-γ
・ Ovalbumin
The following polypeptides were purchased from BACHEM and used.
・ Angiotensin II
Polypeptide consisting of amino acids 323 to 339 of ovalbumin (ovalbumin (323-339))
The following polypeptides were purchased from Calbiochem and used.
Angiotensin II ([Tyr (PO ₃ H ₂ ) ⁴ ] -angiotensin II) in which the fourth amino acid sequence tyrosine was phosphorylated

Equipment Quadrupole tandem MS equipment: API365 (Applied Biosystems)
LC system: LC600 (GL Science)

Preparation of polypeptide stock solution GRF and insulin were dissolved in an acetic acid aqueous solution having a volume ratio of 0.1% to prepare a stock solution (100 μM). Urocortin was dissolved in an acetic acid aqueous solution having a volume ratio of 1% to prepare a urocortin stock solution (100 μM). Amyloid β-protein (1-28), (1-38), (1-40), (1-42) and (1-43) were dissolved in DMSO to prepare a stock solution (100 μM). [Tyr (PO ₃ H ₂ ) ⁴ ] -angiotensin II and ovalbumin (323-339) are dissolved in water to dissolve the polypeptide stock solution (1 mM), and angiotensin II is dissolved in water to dissolve the polypeptide stock solution ( 50 mM) was prepared. Moreover, midkine, CINC-1 / gro, and PTH (1-84) prepared the polypeptide stock solution (10 micromol) by melt | dissolving in water.
Furthermore, ovalbumin was dissolved in water to prepare a polypeptide stock solution (10 mg / mL; about 200 μM). Other polypeptides were dissolved in water to prepare a polypeptide stock solution (100 μM).

Setting monitor ions by measuring polyvalent ions of a polypeptide <Sample preparation>
10 μL of the polypeptide stock solution (100 μM) was mixed with an acetic acid-water-acetonitrile-methanol mixture (volume ratio 2: 80: 10: 10, 4: 80: 10: 10, 2: 50: 25: 25 or 4:50:25). 25) 490 μL or 990 μL, or acetic acid-water-acetonitrile mixture (volume ratio 2:80:20, 4:80:20, 2:50:50 or 4:50:50) added to 490 μL or 990 μL, Peptide sample solutions (1 or 2 μM) were prepared. However, after diluting the [Tyr (PO ₃ H ₂ ) ⁴ ] -angiotensin II and ovalbumin (323-339) stock solution 10 times with water (100 μM), 1 μm or 2 μM sample solution was similarly used using 10 μL thereof. Was prepared. Moreover, after diluting angiotensin II stock solution 50 times with water (1 mM), a sample solution of 1 or 2 μM was prepared using 10 μL thereof. Furthermore, 50 μL of midkine, CINC-1 / gro and PTH (1-84) stock solution (10 μM) were mixed with an acetic acid-water-acetonitrile-methanol mixture (volume ratio 4: 70: 10: 10 or 4: 40: 25: 25). ) To 450 μL to prepare a polypeptide sample solution (1 or 2 μM). 10 μL of Ovalbumin stock solution (10 mg / mL; about 200 μM) is added to 490 μL or 990 μL of acetic acid-water-acetonitrile-methanol mixture (volume ratio 4: 80: 10: 10 or 4: 50: 25: 25), and the polypeptide A sample solution (1 or 2 mg / mL) was prepared.
<Measurement conditions>
Measurement mode: ESI positive
Sample introduction method: Infusion method Flow rate: 5 μL / min
<Monitor ion setting>
Multivalent ions were observed in all the polypeptides used for the study. One of the multivalent ions was selected as a parent ion, and daughter ions were selected for MS / MS measurement. At that time, parameters related to the MS conditions were optimized. Table 1 and Table 2 show examples of monitor ions of each polypeptide used in the measurement.

Example 1 (Phase transition phenomenon of adsorption ability of urocortin to column packing material)
<Sample preparation>
10 μL of urocortin stock solution (100 μM) was added to 90 μL of 2% acetic acid aqueous solution to prepare a urocortin sample solution (10 μM). Further, 10 μL of this urocortin sample solution was added to 990 μL of water-acetonitrile mixed solution (volume ratio of 10: 0, 8: 2, 7: 3, 6: 4, 4: 6 or 2: 8) containing acetic acid at a volume ratio of 4%. The urocortin sample solution (100 nM) was prepared. Further, formic acid having a volume ratio of 4% instead of acetic acid having a volume ratio of 4%, or a water-acetonitrile mixture containing TFA having a volume ratio of 0.1% (volume ratios of 10: 0, 8: 2, 7: 3, 6: 4, 4: 6, 2: 8) and an acid-free water-acetonitrile mixture (volume ratios 10: 0, 8: 2, 7: 3, 6: 4, 4: 6, 2: 8). A urocortin sample (100 nM) with different acetonitrile content was prepared. Furthermore, 1 nM urocortin sample solution was prepared by diluting 10 μL of 100 nM urocortin sample solution with 990 μL of the same composition.

Mobile phase A: Acetic acid-water mixture (volume ratio 4: 100)
Mobile phase B: Acetic acid-acetonitrile-methanol mixture (volume ratio 4:50:50)
Column: _{C 4} reverse phase column (Develosil300C4-HG-5: inner diameter 2.0 mm, length 100 mm, particle size 5 [mu] m)
C ₃₀ reverse phase column (Develosil _C30 -UG-3: inner diameter 2.0 mm, length 100 mm, particle diameter 3 μm)
Column temperature: 50 ° C
Flow rate: 0.2 mL / min
Gradient:

  However, in the system of the present invention, the flow rate was set to 0.6 mL / min from 0.1 minute to 3 minutes.

  500 μL or 100 μL of 1 nM urocortin sample solution with different acetonitrile content and added acid was introduced into the system.

<Result>
Urocortin samples with different concentrations of acetonitrile and different acids in the solution were measured using the reverse phase liquid chromatographic method (FIG. 1 (A)) and _C30 column, which are conventionally used as polypeptide quantification methods. did. As a result, the peak area of urocortin was not constant and changed as shown in FIG. Except for the case where TFA was used as the acid, the peak area of urocortin showed the maximum value when the acetonitrile content in the sample solution was 30%. On the other hand, when TFA was used as the acid, the urocortin peak area showed the maximum value when the acetonitrile content in the sample solution was 40%, but the peak area at that time was the peak when another acid was used. It was almost the same as the area. Therefore, the influence of the acid on the peak area of urocortin was considered to be smaller than the influence of the acetonitrile content in the sample solution.

The above acetonitrile content of different urocortin sample solution (including the volume ratio of 4% acetic acid) was compared with mass chromatograms when measured using a C ₄ column. As a result, when a urocortin sample solution having an acetonitrile content of 40% or more in the solution was measured, in addition to the urocortin peak with a retention time of 6.5 minutes, a new peak was observed at about 1.5 minutes. (FIG. 3). Since the urocortin sample solution contains no test substance other than urocortin, this peak was considered to be urocortin. However, in order for this newly appearing peak to be urocortin from a retention time of about 1.5 minutes, it is necessary for urocortin to pass through the column. Therefore, in order to explain these phenomena, it was hypothesized that the following "phase transition of adsorption ability of urocortin to column packing material" occurred. That is, when the acetonitrile content around urocortin exceeds a certain specific value (which is generally called a critical point or critical value in the phase transition phenomenon, hereinafter referred to as critical point or critical value), urocortin However, if the acetonitrile content around urocortin is less than its critical value, the adsorption capacity of urocortin to the column packing is sufficient to retain the column. It was hypothesized that the adsorption ability of urocortin to the column packing material changes abruptly at the critical point.

  Assuming the above, this phenomenon can be explained as follows. That is, when the acetonitrile content in the urocortin sample solution is 40% or more (exceeding the critical value), the urocortin in the sample solution is present in the solution in a state where the adsorption capacity to the column packing material has been lost (hereinafter, the column A phase of urocortin that does not interact at all with the filler or interacts significantly weakly is sometimes referred to as OFF-phase urocortin). In this state, as shown in FIG. 4, the sample solution introduced into the reverse phase chromatograph maintains its acetonitrile content until it is introduced into the column, so that urocortin in the solution cannot interact with the column. , Pass through the column. On the other hand, when the acetonitrile content is 30% or less (below the critical value), urocortin in the sample solution is present in the solution while maintaining the adsorption ability to the column packing (hereinafter, strongly applied to the column packing). The interacting phase urocortin is sometimes referred to as ON-phase urocortin). In that state, the sample solution introduced into the reverse phase chromatograph maintains its acetonitrile content until it is introduced into the column, as shown in FIG. 4, so that urocortin in the solution interacts with the column, It will be held in the column.

  However, even when the acetonitrile content in the urocortin sample solution is 40% or more (exceeding the critical value), the diffusion of the sample solution that occurs before being introduced into the column (FIG. 4B), that is, the sample solution and It is considered that mixing with the mobile phase occurs before and after the sample solution. A retention time of 6 is obtained because the solution composition around the urocortin (mixed with the sample solution and the mobile phase) generated by the diffusion (particularly the back side of the sample solution) becomes a composition showing the adsorption ability of urocortin to the column filler. It was considered that the result was recognized as a peak of 5 minutes.

  From this result, it is considered that the critical value of acetonitrile content in the sample solution that causes “phase transition of adsorption ability of urocortin to the column packing material” exists between 30% and 40%.

  Based on this hypothesis, the system shown in FIGS. 1 (C) and (D) was devised as a system for polypeptide quantification. When this system is used, the mobile phase B (or eluent consisting of the mobile phase B and the mobile phase C), which is a flow containing the polypeptide sample solution, and the mobile phase A, which is a separate flow, are mixed before introducing the column. Therefore, by changing the mixing ratio of the mobile phases A and B, the polypeptide peripheral solution composition at the column tip can be arbitrarily changed. For example, in FIG. 1C, when mobile phase A is water, mobile phase B is acetonitrile, and the mixing ratio is set to 8: 2, the acetonitrile content at the column tip after mixing is prepared only with acetonitrile. Even when the measured polypeptide sample solution is measured, it remains 20%. On the other hand, when a polypeptide sample solution prepared only with water is measured, the acetonitrile content at the column tip after mixing is temporarily 0%. That is, as long as this mobile phase ratio is maintained, the acetonitrile content at the column tip is only 20% at the maximum regardless of the acetonitrile content in the polypeptide sample solution. Therefore, for example, in the case of urocortin, even if a urocortin sample in a state where the adsorption capacity to the column packing material has been lost (acetonitrile content exceeding a critical value, that is, a solution composition having an acetonitrile content of 40% or more) is introduced into the system, By setting the mobile phase A: B ratio to the ratio A: B that can restore the affinity of urocortin to the column stationary phase, the adsorption ability of urocortin to the column packing can be instantaneously generated by the phase transition, As a result, it was considered that all urocortin could be retained on the column.

To test this hypothesis, again, the acetonitrile content is different for the same concentration urocortin sample solution in the solution (including a volumetric ratio of 4% acetic acid), C ₄ conventionally equipped with a column system diagram 1 (A) and The present system was measured using FIG. 1C (1 nM; injection volume 100 μL). As a result, when the conventional system was used, the urocortin peak area with a retention time of 6.5 minutes changed almost the same as the previous time (FIG. 5B), and the urocortin peak with a retention time of 1.5 minutes was This was observed when the acetonitrile content in the solution was 40% or more, and the area increased as the acetonitrile content increased (FIG. 5 (A) black circle). On the other hand, when the system of the present invention was used, when the acetonitrile content in the urocortin sample solution was 30% or more, the peak area of urocortin was almost constant (FIG. 5 (B), white circle), and was recognized by the conventional method. No urocortin peak corresponding to a retention time of 1.5 minutes was observed in the system of the present invention (FIG. 5 (A), white circles). In addition, when the acetonitrile content in the urocortin sample solution is 20% or less (below the critical value), the urocortin peak area with a retention time of 6.5 minutes is compared with the case where the acetonitrile content in the sample solution is 30% or more. Further, when the acetonitrile content in the sample solution was 0%, the peak area of urocortin was about 1/40. This decrease in the urocortin peak area is due to the adsorption of the urocortin sample into the column by adsorption to the equipment and container (Eppendorf chip and tube) from which the urocortin sample was prepared and the equipment used in the liquid chromatograph (injection syringe). It was thought to be due to the decrease in the amount of Moreover, since the acetonitrile content in the urocortin sample solution was 30% or more and the peak area was almost constant, when the acetonitrile content in the sample solution was 30% or more, the container used for sample preparation and storage and sample introduction It was thought that adsorption to the syringe etc. used occasionally did not occur. From this, by handling urocortin in a solution containing an organic solvent content larger than the phase transition critical value (between 30% and 40%) of the adsorption ability of urocortin to the column packing used this time, It was thought that the loss due to adsorption of urocortin to the container and apparatus during sample introduction could be avoided.

  From this examination result, it was considered that the phase transition of the adsorption ability of urocortin to the column packing was surely taking place. That is, the OFF-phase urocortin introduced into the system of the present invention instantaneously undergoes a phase transition in the solution generated by mixing with the aqueous mobile phase to become the ON-phase urocortin, and all urocortin can interact with the column. It was considered that almost the same peak area was detected when adsorption to the container and syringe was not observed before the sample was introduced. In addition, it is possible to roughly grasp the acetonitrile content that prevents adsorption to containers and devices, etc. by grasping the phase transition critical value of the adsorption capacity to the column packing material using a silica gel-supported column as used this time. It was suggested.

Example 2 (Phase transition of adsorption ability of urocortin to column filler by factors other than acetonitrile)
<Sample preparation>
10 μL of urocortin stock solution (100 μM) was added to 990 μL of an acetic acid-water-acetonitrile mixture (volume ratio 4:50:50) to prepare a urocortin sample solution (1 μM). Furthermore, 10 μL of this urocortin sample solution was added to 990 μL of the following mixed solution to prepare a urocortin sample solution (10 nM).
Water-acetonitrile mixture (volume ratio = 7: 3, 6: 4, 5: 5, 4: 6, 3: 7, 2: 8 or 1: 9)
Water-ethanol mixture (volume ratio = 7: 3, 6: 4, 5: 5, 4: 6, 3: 7, 2: 8 or 1: 9)
Water-methanol mixture (volume ratio = 7: 3, 6: 4, 5: 5, 4: 6, 3: 7, 2: 8 or 1: 9)
Water-acetic acid mixture (volume ratio = 7: 3, 6: 4, 5: 5, 4: 6, 3: 7, 2: 8 or 1: 9)
Water-formic acid mixture (volume ratio = 7: 3, 6: 4, 5: 5, 4: 6, 3: 7, 2: 8 or 1: 9)

<Measurement conditions>
Mobile phase A: Acetic acid-water mixture (volume ratio 4: 100)
Mobile phase B: Acetic acid-acetonitrile-methanol mixture (volume ratio 4:50:50)
Column: _{C 4} reverse phase column (Develosil300C4-HG-5: inner diameter 2.0 mm, length 100 mm, particle size 5 [mu] m)
Column temperature: 50 ° C
Flow rate: 0.2 mL / min
Gradient:

  100 μL of 10 nM of each urocortin sample solution was introduced into the system.

<Result>
It was shown that the phase transition of adsorption capacity of urocortin to the column packing was caused by all the solutions studied, not just acetonitrile as described above (Table 5). The phase transition critical value in each solution is 40% to 50% by volume when ethanol is used, 60 to 70% by volume when using methanol and acetic acid, and 80% to 90% by volume when using formic acid. % Were considered to be present. From this result, it was shown that the strength of the organic solvent contained in the solution that gave the phase transition of the adsorption ability of urocortin to the column packing was in the order of acetonitrile>ethanol> methanol and acetic acid> formic acid. However, when a high concentration of formic acid was used, the phenomenon that the peak area of urocortin decreased with time was observed, and it was considered that the formylation of the amino group present in urocortin was caused. From this, it was considered that it is not preferable to use a high concentration of formic acid in the sample.

Example 3 (Phase transition phenomenon of adsorption ability of urocortin in a solution containing two organic solvents to a column packing material)
<Sample preparation>
10 μL of urocortin stock solution (100 μM) was added to 990 μL of an acetic acid-water-acetonitrile mixture (volume ratio 4:50:50) to prepare a urocortin sample solution (1 μM). Further, 10 μL of this urocortin sample solution was added to 990 μL of the mixed solution shown in Tables 6 and 7, to prepare a urocortin sample solution (10 nM).

<Measurement conditions>
Mobile phase A: Acetic acid-water mixture (volume ratio 4: 100)
Mobile phase B: Acetic acid-acetonitrile-methanol mixture (volume ratio 4:50:50)
Column: _{C 4} reverse phase column (Develosil300C4-HG-5: inner diameter 2.0 mm, length 100 mm, particle size 5 [mu] m)
Column temperature: 50 ° C
Flow rate: 0.2 mL / min
Gradient:

  100 μL of each polypeptide mixed sample solution was introduced into the system.

<Result>
Tables 9 and 10 show the peak area values of urocortin that were passed through without being held on the column when a urocortin sample containing two kinds of organic solvents was measured using a conventional method.

  From this result, it was considered that the phase transition phenomenon of the adsorption ability of urocortin to the column filler in a mixed solution containing two or more organic solvents follows the following formula (a). Actually, values f calculated by substituting the respective organic solvent contents used in the urocortin sample into the formula (a) are shown in Tables 11 and 12, and before and after f becomes 1, the urocortin column filler is obtained. It was confirmed that a phase transition of the adsorption capacity of However, the critical content (%) of each organic solvent used in the calculation is an intermediate value of the organic solvent content before and after the peak is divided into two on the chromatogram (acetonitrile: 35%, ethanol: 45%, methanol: 65% Acetic acid: 65%, formic acid: 85%).

  Expression (a) representing the relationship between the phase transition phenomenon of the adsorption ability of polypeptide A to the column filler and the content of each organic solvent in a sample containing two or more organic solvents

Under certain conditions,
X: volume% showing the critical value of phase transition of polypeptide A in water-organic solvent A mixed solution
Y:% by volume indicating the critical value of phase transition of polypeptide A in water-organic solvent B mixed solution
Z: Volume% showing the phase transition critical value of polypeptide A in water-organic solvent C mixed solution
x: organic solvent A content% in the sample solution
y: organic solvent B content% in the sample solution
z: organic solvent C content% in the sample solution

Example 4 (Phase transition phenomenon of adsorption ability of urocortin in a solution containing three organic solvents to a column packing material)
<Sample preparation>
10 μL of urocortin stock solution (100 μM) was added to 990 μL of an acetic acid-water-acetonitrile mixture (volume ratio 4:50:50) to prepare a urocortin sample solution (1 μM). Furthermore, 10 μL of this urocortin sample solution was added to 990 μL of the mixed solution shown in Table 13 to prepare a urocortin sample solution (10 nM).

<Measurement conditions>
Mobile phase A: Acetic acid-water mixture (volume ratio 4: 100)
Mobile phase B: Acetic acid-acetonitrile-methanol mixture (volume ratio 4:50:50)
Column: _{C 4} reverse phase column (Develosil300C4-HG-5: inner diameter 2.0 mm, length 100 mm, particle size 5 [mu] m)
Column temperature: 50 ° C
Flow rate: 0.2 mL / min
Gradient:

  100 μL of each polypeptide mixed sample solution was introduced into the system.

<Result>
When a urocortin sample containing three organic solvents (acetonitrile, acetic acid, and methanol) was measured using a conventional method, the peak area value of urocortin that was passed through without being held in the column, and the value f calculated from Equation (1) f Is shown in Table 15. Similar to Example 3, this result also suggested that the phase transition of the adsorption ability of urocortin to the column filler in the sample containing a plurality of organic solvents follows the formula (a). Based on the above results, the phase transition critical value (volume%) of the adsorption ability of urocortin to the column packing when using a mixed solvent of water and each organic solvent is known in advance. It was shown that it is possible to predict the adsorption ability (ON phase or OFF phase state) of urocortin to a column packing material in a sample containing a plurality of organic solvents.

Example 5 (Phase transition of adsorption ability of various polypeptides to C4 column packing material by each organic solvent (including organic acid))
<Sample preparation>
Among all 27 types of polypeptides used in the study, 10 μL of 25 types of polypeptide stock solutions (1 mM, 100 μM, 10 μM, or 10 mg / mL) excluding angiotensin II and ovalbumin (323-339) were respectively 990 μL. Each polypeptide sample solution (10 μM, 1 μM, 100 nM or 0.1 mg / mL) was prepared.
Water-acetonitrile mixture (volume ratio = 95: 5, 9: 1, 8: 2, 7: 3, 6: 4, 5: 5, 4: 6 or 3: 7)
Water-ethanol mixture (volume ratio = 95: 5, 9: 1, 8: 2, 7: 3, 6: 4, 5: 5, 4: 6 or 3: 7)
Water-methanol mixture (volume ratio = 95: 5, 9: 1, 8: 2, 7: 3, 6: 4, 5: 5, 4: 6 or 3: 7)
Water-acetic acid mixture (volume ratio = 95: 5, 9: 1, 8: 2, 7: 3, 6: 4, 5: 5, 4: 6 or 3: 7)
Also, 990 μL of the above solution (10 μL) obtained by diluting ovalbumin (323-339) stock solution (1 mM) 10 times with water (100 μM) and angiotensin II stock solution (50 mM) 50 times diluted with water (990 μL) Each polypeptide sample solution (1 μM or 10 μM) was prepared by adding to the solution.

Mobile phase A: Acetic acid-water mixture (volume ratio 4: 100)
Mobile phase B: Acetic acid-acetonitrile-methanol mixture (volume ratio 4:50:50)
Column: _{C 4} reverse phase column (Develosil300C4-HG-5: inner diameter 2.0 mm, length 100 mm, particle size 5 [mu] m)
Column temperature: 50 ° C
Flow rate: 0.2 mL / min
Gradient:

  100 μL of 1 μM polypeptide mixed sample solution was introduced into the system.

<Result>
The phase transition of the adsorption ability to the column packing observed when each polypeptide solution is measured using the conventional system (FIG. 1A), that is, the phenomenon that the peak of the polypeptide to be measured is divided into two. It was observed in all polypeptides used in the study. Table 17 shows the maximum organic solvent (including acetic acid) content in the solution in which each polypeptide is recognized as a single peak on the chromatogram (the phase transition critical value of the adsorption capacity to the column packing is slightly different from this value). It is thought that it exceeds.) However, in this study, even when a water-organic solvent mixture (volume ratio = 95: 5) is measured, if it is not recognized as one peak on the chromatogram, it is expressed as 5% or less (<5%). did.
From the present results, the strength of the organic solvent causing the phase transition of the adsorption ability of the polypeptide to the column packing material was almost the same for each polypeptide, and was almost the same for acetonitrile and ethanol, followed by methanol. On the other hand, it was suggested that the strength of the effect of acetic acid, which is an organic acid, on the phase transition of the adsorption ability of each polypeptide to the column packing material is almost the same as or slightly weaker than that of methanol. In addition, since a positive correlation was observed between the maximum organic solvent content in these solutions and the retention time of each polypeptide, the polypeptide was also affected by organic solvents such as acetonitrile and organic acids contained in the eluent. It was expected to cause a phase transition of adsorption capacity. Therefore, it was considered that the polypeptide retained in the column was eluted while repeating the adsorption ability phase transition caused by the organic solvent contained in the eluent, that is, adsorption and desorption on the column packing material.

Example 6 (Retention time of each polypeptide and power gradient gradient gradient)
<Sample preparation>
10 μL of each polypeptide (18 kinds of polypeptides in Table 1) stock solution (100 μM) was added to 820 μL of acetic acid-water (volume ratio 4: 100) to prepare a polypeptide mixed sample solution (1 μM each). . Furthermore, by adding 9 other kinds of polypeptide stock solutions to acetic acid-water (volume ratio 4: 100), another polypeptide mixed sample solution (each 1 μM) is prepared so that the concentration of each polypeptide becomes 1 μM. did. However, the concentration of ovalbumin was adjusted to 1 mg / mL.

Mobile phase A: acetic acid-water (volume ratio 4: 100)
Mobile phase B: Acetic acid-acetonitrile-methanol mixture (volume ratio 4:50:50)
Column: _{C 4} reverse phase column (Develosil300C4-HG-5: inner diameter 2.0 mm, length 100 mm, particle size 5 [mu] m)
Column temperature: 50 ° C
Flow rate: 0.2 mL / min
Gradient:

  10 μL of 1 μM polypeptide mixed sample solution was introduced into the system.

<Result>
As a result of measuring the retention time of the polypeptide when the gradient gradient was changed to 0.5, 1, 2, 4, 6, 8, 10% / min by the conventional method (FIG. 1A), A power law was observed between the retention time of the retained polypeptide and the gradient gradient (except for nocetictin and amyloid β-protein (1-16), which are hardly retained in the column under the present measurement conditions) ). As an example, the results for urocortin are shown in FIG. The characteristic of the polypeptide eluted according to this power law is that the retention time is infinite when the gradient gradient is as close as possible to 0% / min (when moving to the left on the x-axis in FIG. 6). That is, the polypeptide is not eluted.
Next, assuming that polypeptide A is eluted by the “phase transition of adsorption capacity to the column packing material” under the present measurement conditions, elution at the moment when polypeptide A is eluted regardless of the gradient gradient. Since the composition of the organic solvent in the liquid is constant, that is, the ratio of the mobile phases A and B constituting the eluent at the moment when the polypeptide A is eluted is considered to be constant. It is considered that equations (3) and (b) regarding the ratio% (V _B ) of the mobile phase B constituting the eluent at the moment when A is eluted are established.
In this study, using the retention time of each polypeptide obtained when the gradient gradient is 4% / min, 6% / min, and 8% / min, the dead volume of the entire measurement system is calculated from equation (3). The calculated results are shown in Table 19. Regardless of the gradient slope, it was shown that the dead volume of each polypeptide was constant at approximately 3 minutes and was not dependent on the polypeptide. In addition, a tendency that the dead volume slightly increases as the molecular weight decreases is considered to be due to the fact that these peptides can reach the inside of the column pores more.
Subsequently, for the 27 types of polypeptides used in the examination of Example 5, the percentage (V _B ) of the mobile phase B constituting the eluent at the moment when each polypeptide was eluted was calculated from the formula (b). Thereafter, the content of each organic solvent contained in the eluent at the moment when each polypeptide is eluted is calculated, and the value f calculated by substituting the content of each organic solvent into the formula (a) is calculated. Shown in The critical organic solvent content (%) used in the calculation was an intermediate value between the maximum organic solvent content (%) lower than the critical value in Example 5 and the minimum organic solvent content (%) at which the peak was split into two. . However, when the maximum organic solvent content (%) below the critical value was 5% or less, it was set as 5% as it was. As a result of the calculation, it was shown that each polypeptide was present and eluted in the eluent where f was approximately 1. Among the peptides, there was a case where the f value was significantly different from 1.30 to 2.03, but most of these had a maximum organic solvent content (%) lower than the critical value in Example 5. This is a polypeptide showing 5% or less, which is considered to be due to the large influence of the difference of 1% in the critical organic solvent content (%) used in the formula (a).
From the above results, the polypeptide is generally eluted from the column by the “phase transition of the adsorption ability of the polypeptide to the column filler” caused by each organic solvent contained in the eluent. The relationship between the phase transition of the adsorption capacity to the packing material and each organic solvent contained in the eluent is shown in Example 3 “Adsorption capacity of polypeptide in column packing material in a sample containing a plurality of organic solvents”. It was suggested that this is the same as the formula (a) "representing the relationship between the phase transition and the content of each organic solvent.

When polypeptide A is measured using a conventional system and two mobile phases (aqueous mobile phase A and organic solvent mobile phase B), polypeptide A is eluted by “phase transition of adsorption capacity to column packing material”. Assuming that the total organic solvent content in the eluent at the instant when polypeptide A is eluted is constant regardless of the gradient gradient, that is, the mobile phase constituting the eluent at the instant when polypeptide A is eluted Since the ratio ratio of A and B is considered to be constant, the following equation is established with respect to the ratio% (V _B ) of the mobile phase B constituting the eluent at the instant when the polypeptide A is eluted.
V ₁ = C _b + (T ₁ −t ₀ ) · r ₁
V ₂ = C _b + (T ₂ −t ₀ ) · r ₂
C _b : percentage of mobile phase B under initial measurement conditions
r ₁ and r ₂ : gradient gradient (% / min)
T _1: Gradient gradient r ₁ when measured in a polypeptide retention time of A T _2: gradient gradient r ₂ the retention time of polypeptide A when measured at t _0: the dead volume (porosity columns and HPLC system Value obtained by dividing the sum of voids ahead of the injector by the flow velocity) (minutes)
If V ₁ = V ₂ ,
Dead volume _{t 0} is,

Furthermore, the ratio% (V _B ) of the mobile phase B constituting the eluent at the instant when the polypeptide A is eluted is the ratio% of the initial organic solvent mobile phase B C _b = 0,
V _B = (T ₁ −t ₀ ) · r ₁
V _B = (T ₂ −t ₀ ) · r ₂
2V _B = (T ₁ −t ₀ ) · r ₁ + (T ₂ −t ₀ ) · r ₂
And substituting dead volume t ₀ (formula (3)) for

It becomes.

In addition, the power between the retention time of the polypeptide held in the column and the gradient gradient, which is observed when the gradient gradient is changed to 0.5, 1, 2, 4, 6, 8, 10% / min. The constant B obtained from the approximate curve of the function y = Bx− ^t (y: retention time, x: gradient gradient, t: power exponent, where t> 0) is shown in Table 21 (however, under the present measurement conditions) Noceptin and amyloid β-protein (1-16) which are hardly retained in the column). When the gradient gradient is 1% / min (x = 1), y = B regardless of the power index t. Therefore, it is clear that the constant B indicates the retention time when the gradient gradient is 1% / min. Furthermore, the retention time in the case of a gradient gradient of 1% / min is considered to be equivalent to the organic solvent volume (retention time × gradient gradient) increased in the eluent until the polypeptide is eluted (retention time × gradient gradient). However, dead volume is included). Under the conditions of this study, the proportion of the organic solvent at the start of measurement is only 4% by volume of acetic acid contained in the aqueous mobile phase A, and the dead volume of the entire measurement system is about 3 minutes. From the above, the obtained constant B is an approximate value of the organic solvent content which is the phase transition critical value of each polypeptide (it is considered that there is a difference of about 1 minute which is the difference between the acetic acid content of 4% and the dead volume). It was thought to show. Therefore, when compared with the ratio% (V _B ) of the mobile phase B constituting the eluent at the moment when the polypeptide A was eluted, a substantially identical result was obtained.
Therefore, from this result, the retention time obtained by measuring the organic solvent content at the start of the measurement at 0% and the gradient gradient at 1% / min is the value of the organic solvent content that is the phase transition critical value of each polypeptide. It was shown to show an approximate value. Therefore, in determining the critical value (content) of the phase transition caused by the single organic solvent of each polypeptide, the phenomenon that the peak of the polypeptide to be measured is divided into two as in Examples 1 to 5 is confirmed. Even if such a complicated measurement is not performed, it is possible to obtain an approximate value of the phase transition critical value of each polypeptide by performing a single measurement using a single organic solvent as the organic solvent-based mobile phase. it was thought. With this measurement method, it was possible to measure a sample to which a plurality of polypeptides were added, and as a result, it was considered possible to simultaneously measure the phase transition critical values of the plurality of polypeptides.

Example 7 (Relationship between retention time of each polypeptide and phase transition critical value when measured at a gradient gradient of 1% / min)
<Sample preparation>
10 μL of each polypeptide (18 kinds of polypeptides in Table 1) stock solution (100 μM) was added to 820 μL of an acetic acid-water mixture (volume ratio 4: 100), and a polypeptide mixed sample solution (1 μM each) was added. Prepared. Furthermore, by adding nine other kinds of polypeptide stock solutions to acetic acid-water (volume ratio 4: 100), another polypeptide mixed solution (each 1 μM) was prepared so that the concentration of each polypeptide was 1 μM. . However, the concentration of ovalbumin was adjusted to 1 mg / mL.

Mobile phase A: acetic acid-water (volume ratio 4: 100)
Mobile phase B: Acetic acid-acetonitrile mixture (volume ratio 4: 100), acetic acid-methanol mixture (volume ratio 4: 100), acetic acid-ethanol mixture (volume ratio 4: 100), or 100% acetic acid column: C ₄ reverse phase column (Develosil 300C4-HG-5: inner diameter 2.0 mm, length 100 mm, particle diameter 5 μm)
Column temperature: 50 ° C
Flow rate: 0.2 mL / min
Gradient:

  10 μL of 1 μM polypeptide mixed sample solution was introduced into the system.

<Result>
Using the conventional method (FIG. 1 (A)), each retention time at a gradient gradient of 1% / min obtained under the measurement conditions used this time is the phase transition criticality of each polypeptide as described in Example 6. It was suggested to show an approximate value of the content of organic solvent. Therefore, the type of organic solvent in mobile phase B was changed, and the retention time indicated by each polypeptide at a gradient gradient of 1% / min was estimated from the phase transition criticality estimated from the phenomenon that the peak was divided into two in Example 5. When compared with the value range, as expected, most of the obtained retention time was within the estimated critical value range, indicating almost the same tendency (Table 23). Therefore, it was suggested that the adsorption ability phase transition caused by the change in the organic solvent content in the polypeptide solution and the adsorption ability phase change caused by the change in the organic solvent content in the eluent are the same.
In this study, since the aqueous mobile phase contains acetic acid with a volume ratio of about 4% at the start of measurement and the detected retention time includes a dead volume of about 3 minutes, In the case of a short polypeptide, a little care is required in handling the obtained value, but the method of estimating the critical value shown by each organic solvent from the retention time under the measurement conditions as used this time is In terms of sample preparation and the like, it was considered to be simpler and more accurate than the method of estimating from the phenomenon that the peak splits into two.

Example 8 (Influence of column stationary phase on polypeptide retention time)
<Sample preparation>
10 μL of each polypeptide (18 kinds of polypeptides in Table 1) stock solution (100 μM) is added to 820 μL of an acetic acid-water mixture (volume ratio 4: 100), and a polypeptide mixed sample solution (each 1 μM) is added. Prepared.

Mobile phase A: acetic acid-water (volume ratio 4: 100)
Mobile phase B: Acetic acid-acetonitrile-methanol mixture (volume ratio 4:50:50)
Column: C ₄ , C ₈ , C ₁₈ and C ₃₀ reverse phase column (Develosil 300C4-HG-5: inner diameter 2.0 mm, length 100 mm, particle diameter 5 μm)
(Develosil 300C8-HG-5: inner diameter 2.0 mm, length 100 mm, particle diameter 5 μm)
(Develosil 300ODS-HG-5: Inner diameter 2.0 mm, Length 100 mm, Particle diameter 5 μm)
(Develosil RPAQUEOUS-AR-3: inner diameter 2.0 mm, length 100 mm, particle diameter 3 μm)
Column temperature: 50 ° C
Flow rate: 0.2 mL / min
Gradient:

  10 μL of 1 μM polypeptide mixed sample solution was introduced into the system.

<Result>
Results of examining the influence of the column stationary phase to give the retention time of each polypeptide, the polypeptide showed the shortest retention time in the case of using the C ₄ column, substantially constant hold regardless of the column stationary phase It was shown to have time (Table 25). The results show that elution of the polypeptide is an organic solvent (including organic acids) such as acetonitrile contained in the mobile phase, rather than the hydrophobic interaction with the column stationary phase, which is the main separation factor for low molecular weight compounds. It was suggested that it was greatly influenced by the “phase transition of the adsorption ability of the polypeptide to the column packing” caused by

Example 9 (Influence of column temperature on retention time of polypeptide)
<Sample preparation>
10 μL of each polypeptide (18 kinds of polypeptides in Table 1) stock solution (100 μM) is added to 820 μL of an acetic acid-water mixture (volume ratio 4: 100), and a polypeptide mixed sample solution (each 1 μM) is added. Prepared.

<Measurement conditions>
Mobile phase A: acetic acid-water (volume ratio 4: 100)
Mobile phase B: Acetic acid-acetonitrile-methanol mixture (volume ratio 4:50:50)
Column: _{C 4} reverse phase column (Develosil300C4-HG-5: inner diameter 2.0 mm, length 100 mm, particle size 5 [mu] m)
Column temperature: 20, 30, 40 or 50 ° C
Flow rate: 0.2 mL / min
Gradient:

  10 μL of 1 μM polypeptide mixed sample solution was introduced into the system.

<Result>
In general, in the measurement of a low molecular weight compound, as the column temperature is lowered, the hydrophobic interaction between the low molecular weight compound and the column stationary phase becomes stronger, and the retention time becomes considerably longer. However, each polypeptide used in this study showed a longer retention time at a lower column temperature, but the difference was small in most cases (Table 27). From this result, elution of polypeptide is greatly influenced by “phase transition of adsorption ability of polypeptide to column packing material” caused by organic solvent (including organic acid) such as acetonitrile contained in mobile phase. It was thought to receive. On the other hand, it was thought that changing the column temperature could also cause the “phase transition of the adsorption capacity of the polypeptide to the column packing material”, but the organic solvent such as acetonitrile in the sample solution or mobile phase was It was considered to be extremely small compared to the effect.

Example 10 (Accuracy comparison between conventional system and system of the present invention when measuring urocortin sample)
<Sample preparation>
10 μL of urocortin stock solution (100 μM) was added to 990 μL of an acetic acid-water-acetonitrile mixture (volume ratio 4:50:50) to prepare a urocortin sample solution (1 μM). Further, 10 μL of this urocortin sample solution was added to 990 μL of an acetic acid-water-acetonitrile mixture (volume ratio 4: 100: 0, 4:80:20, 4:70:30, 4:60:40, 4:50:50). 4:40:60 or 4:20:80) to prepare a urocortin sample solution (10 nM). Furthermore, a 10-fold diluted urocortin sample solution (0.1 nM and 1 nM) was prepared using a water-acetonitrile mixture having the same composition.

<Measurement conditions>
Mobile phase A: acetic acid-water (volume ratio 4: 100)
Mobile phase B: Acetic acid-acetonitrile-methanol mixture (volume ratio 4:50:50)
Mobile phase C: acetic acid column: C ₃₀ reverse phase column (RPAQUEOUS-AR-3: inner diameter 2.0 mm, length 35 mm, particle diameter 3 μm)
Column temperature: 50 ° C
Flow rate: 0.2 mL / min
Gradient:

  When using the new method, each mobile phase composition, the mixing ratio at the start of measurement of the organic solvent-based mobile phase (mixed solution of mobile phases B and C) that forms the mobile phase in the line into which the polypeptide is introduced, and From the organic solvent content in the sample containing each polypeptide, the ratio (α) of the aqueous mobile phase to the organic solvent mobile phase mixed in the mixer was calculated based on the aforementioned formula (a) (Table 29). . However, the retention time obtained in Example 7 was used as the phase transition critical value of urocortin in water and each organic solvent. In this study, the ratio of the aqueous mobile phase until the sample containing urocortin was introduced into the column was about 90% (Table 28), which was larger than 52% or 57% shown in Table 29. It was determined that all urocortin introduced into the system was retained on the column because it had a proportion.

  100 μL or 400 μL of 0.1 nM, 1 nM and 10 nM urocortin sample solutions with different acetonitrile contents were introduced into the system. In order to compare the accuracy of the two systems, urocortin sample solutions having the same composition and concentration were measured three times in total, and the average value, standard deviation, and coefficient of variation (%) of the obtained peak area were calculated (daily fluctuation).

<Result>
Tables 30, 31 and 32 show the peak area, standard deviation and coefficient of variation (%) when urocortin samples of the same concentration (0.1 nM, 1 nM and 10 nM) were measured three times by the conventional method and the present invention method.

  When the conventional method was used, the variation coefficient (%) of the peak area of urocortin was 15% or more in most cases regardless of the injection amount and the urocortin concentration, and the variation was large. On the other hand, when the method of the present invention is used, the coefficient of variation (%) of the peak area of urocortin contained in the sample solution containing acetonitrile having a volume ratio of 30% or more is within 15% regardless of the urocortin concentration and the injection amount. In this concentration range, it was considered that adsorption to a container or the like did not occur, or that the concentration range examined did not cause a problem. When the acetonitrile content in the sample solution was 0% and 20% in volume ratio, the coefficient of variation% often showed 15% or more, suggesting a variation considered to be due to adsorption to the container, injection syringe, and the like.

  From the above results, it is considered that the adsorption capacity of urocortin to the container and the syringe for injection is weaker than the phase transition critical value of the adsorption capacity of urocortin to the column packing material. The peak area of urocortin contained in a sample solution having an acetonitrile content exceeding the critical value of phase transition of the present invention is less varied in the method of the present invention than the conventional method, and it is considered that the method of the present invention is superior in terms of accuracy. It was.

  Next, Table 33 shows the peak area ratio (400 μL / 100 μL) obtained when the injection amount of the urocortin sample solution into the LC system was 100 μL and 400 μL.

  When a sample solution containing acetonitrile having a volume ratio of 30% or more, which is considered to cause no adsorption to a container or the like under the present conditions, is measured using a conventional method, the peak area ratio that theoretically shows 4 is 1. A value of 0 to 6.6 was shown, and a large variation from the theoretical value was recognized.

  On the other hand, when a sample solution containing acetonitrile having a volume ratio of 30% or more that is considered not to be adsorbed to a container or the like is measured by the method of the present invention, it is 3.5 to 4.1 regardless of the acetonitrile content and the urocortin concentration. The result was almost proportional to the injection amount. When the acetonitrile content in the sample solution was 0 or 20% by volume, the peak area ratio showed a value of 1.0 to 3.9 even when the method of the present invention was used, and a large variation was recognized.

  From the above results, both the conventional method and the method of the present invention showed the possibility of increasing the sensitivity by increasing the injection amount. However, in the conventional method, the rate of increase is affected by the acetonitrile content in the sample solution. It was done. Moreover, the peak area / height ratio when a 0.1 nM urocortin sample was measured by the conventional method tended to increase as the injection amount increased. Since the peak area / height ratio is considered to correspond to the peak width at half the peak height, assuming that the peak shape of urocortin is a triangle, this result indicates that the peak width of urocortin is widened. (Table 34). Therefore, with the conventional method, it was considered difficult to increase the sensitivity in proportion to the increase in the injection amount. On the other hand, when the sample solution containing acetonitrile (acetonitrile content not causing adsorption to a container or the like) having a volume ratio of 30% or more was measured using the method of the present invention, an increase in peak area proportional to the injection amount was observed. At this time, since the peak area / height ratio is almost constant regardless of the injection amount, it is possible to increase the injection amount to increase the sensitivity in proportion to the injection amount. It was done. In addition, since this high sensitivity is possible regardless of the acetonitrile content in the sample solution, even if the phase transition critical value of the adsorption capacity to the column packing material is influenced by factors other than acetonitrile, the method of the present invention is used. Since the peak area of urocortin is expected to be kept constant by using, it was shown that the system of the present invention is more robust than the conventional method. Furthermore, this result is considered to support that the amount of sample injection when using the new method, which is predicted from the phase transition theory, is unlimited (when injection time and column load are not considered). .

Example 11 (Comparison of both systems in the preparation of a calibration curve for urocortin)
<Sample preparation>
Calibration curve samples with urocortin concentrations of 10, 30, 100, 300 pM, 1, 3 and 10 nM were mixed with 990 μL of an acetic acid-water-acetonitrile mixture (volume ratio 4: 100: 0, 4:80:20, 4:70: 30, 4:60:40, 4:50:50, 4:40:60 or 4:20:80).

<Measurement conditions>
Mobile phase A: acetic acid-water (volume ratio 4: 100)
Mobile phase B: Acetic acid-acetonitrile-methanol mixture (volume ratio 4:50:50)
Mobile phase C: acetic acid column: C ₃₀ reverse phase column (RPAQUEOUS-AR-3: inner diameter 2.0 mm, length 35 mm, particle diameter 3 μm)
Column temperature: 50 ° C
Flow rate: 0.2 mL / min
Gradient:

  When using the new method, each mobile phase composition, the mixing ratio at the start of measurement of the organic solvent-based mobile phase (mixed solution of mobile phases B and C) that forms the mobile phase in the line into which the polypeptide is introduced, and From the organic solvent content in the sample containing each polypeptide, the ratio (α) of the aqueous mobile phase to the organic solvent mobile phase mixed in the mixer was calculated according to the aforementioned equation (a) (Table 36). However, the retention time obtained in Example 7 was used as the phase transition critical value for each organic solvent of urocortin. In this examination, the ratio of the aqueous mobile phase until the sample containing urocortin is introduced into the column is about 90% (Table 35), which is larger than 47% or 57% shown in Table 36. It was determined that all urocortin introduced into the system was retained on the column because it had a proportion.

However, in this development system used this time, the flow rate was set to 0.6 mL / min from 0.1 minute to 8 minutes.
Using the method of the present invention and the conventional method, 10, 30, 100, 300 pM, 1, 3 and 10 nM calibration curve samples were measured, and calibration curves were prepared. The calibration curve was used to create a calibration curve by obtaining the accuracy of the value obtained by backing-calculating each point of the calibration curve with its own calibration curve in accordance with the criteria of the low molecular weight compound validation method (Non-Patent Document 4). The standard for preparing the calibration curve was that the calibration curve sample of 75% or more of the number of calibration curve samples was within ± 20% at the lower limit of quantification and within ± 15% at the other. However, the lower limit of quantification and the upper limit of quantification of the calibration curve must meet the standards. The weight of the calibration curve was set to 1 / square of the concentration, and a calibration curve having the lowest lower limit of quantification was created. The calculation method of the accuracy is as follows.

<Result>
The calibration curves prepared by the conventional method and the method of the present invention are shown in Table 37 and Table 38.

  As a result, when the conventional method was used, a calibration curve satisfying the standard could be prepared except when the acetonitrile content in the sample solution was 50% (Table 37). The lower limit of quantification obtained at this time was 30 pM to 300 pM. However, when a urocortin sample with an acetonitrile content of 0% was measured, there were only four concentrations that could be used as a calibration curve, and in addition, one point whose accuracy did not meet the standard was included in the calibration curve. In many cases, it was suggested that it was difficult to prepare a calibration curve using the conventional method. On the other hand, when the method of the present invention is used, a calibration curve that satisfies the standard created by measuring a urocortin sample having an acetonitrile content of 30% or more by volume has a wide concentration range of 10 pM to 10 nM. All accuracy was within ± 15% (Table 38). In addition, since the slope of the obtained calibration curve showed almost the same value, it was considered that the method of the present invention can be quantified with higher sensitivity and accuracy than the conventional method.

  In addition, as shown in FIG. 7, the peak shape of urocortin when measured using the conventional method was a sharp peak when the acetonitrile content in the sample solution was 30% or more. When a sample solution containing 40% or more of acetonitrile was measured, the peak was leading and it was observed that the peak was broad in the vicinity of the baseline. On the other hand, the peak shape of urocortin measured using the method of the present invention is a sharply rising peak regardless of the acetonitrile content in the sample solution, and this difference in peak shape also contributes to the accuracy when preparing the calibration curve. It was thought to have influenced.

  From the above results, it was considered that the polypeptide of the present invention can be quantified with high accuracy by measuring a sample containing acetonitrile that does not cause adsorption to a container or the like. Moreover, when the method of the present invention was used due to its high accuracy, it was considered that more sensitive quantification (low quantification limit of calibration curve) was possible.

Example 12 (Preparation of polypeptide calibration curve using the system of the present invention)
<Sample preparation>
Among 18 kinds of polypeptides in Table 1, amyloid β-protein (1-16), amyloid β-protein (1-28), amyloid β-protein (1-38), amyloid β-protein (1-42) , 10 μL of 12 polypeptide stock solutions (100 μM) excluding amyloid β-protein (1-43) and insulin are added to 990 μL of an acetic acid-water-acetonitrile mixture (volume ratio 4:50:50). A sample solution (1 μM) was prepared. Furthermore, 10 μL of each polypeptide sample solution was added to 990 μL of a water-acetonitrile mixture (volume ratio 1: 1) containing 4% acetic acid at a volume ratio of 990 μL to prepare each polypeptide sample solution (10 nM). . Furthermore, calibration curve samples of 10, 30, 100, 300 pM, 1, and 3 nM were prepared using solutions of the same composition.

<Measurement conditions>
Mobile phase A: Acetic acid-water mixture (volume ratio 4: 100)
Mobile phase B: Acetic acid-acetonitrile-methanol mixture (volume ratio 70:15:15)
Column: _C30 reverse phase column (RPAQUEOUS-AR-5: inner diameter 2.0 mm, length 35 mm, particle diameter 5 μm)
Column temperature: 50 ° C
Flow rate: 0.2 mL / min
Gradient:

  When using the new method, the ratio (α) of the aqueous mobile phase to the organic solvent mobile phase mixed in the mixer is determined from the composition of each mobile phase and the content of the organic solvent in the sample containing each polypeptide. Calculations were made according to equation (a) (Table 40). However, the retention time obtained in Example 7 (examination using C4 column) was used for the phase transition critical value of each polypeptide for each organic solvent.

  100 μL of each polypeptide sample solution was introduced into the development system. The calibration curve is determined according to the criteria of the validation guidelines, and the accuracy of the value obtained by back-calculating each point of the calibration curve with its own calibration curve is obtained, and the calibration curve is within ± 15% at the lower limit of quantification and within ± 15% at other points It was created. However, 75% or more of the points used for the calibration curve satisfy the standard, and the lower limit of quantification and the upper limit of quantification always meet the standard. The weight of the calibration curve was set to 1 / square of the density.

<Result>
When each polypeptide sample was actually measured, a calibration curve could not be prepared for the polypeptide consisting of amino acids 60 to 121 of nociceptin and midkine. However, for other polypeptides, the lower limit of quantification was 10 pM. Alternatively, a calibration curve of 30 pM could be created (Table 41). The accuracy of the sample concentration obtained from each calibration curve satisfied the standard of ± 15% (lower limit of quantification ± 20%) described in the previous section. The chromatogram at the lower limit of quantification of each polypeptide is shown in FIGS. 8-1 and 8-2. Nociceptin and midkine amino acid sequence 60th to 121st polypeptide with a minimum difference between 94% and the percentage of aqueous mobile phase required to retain in the column Other polypeptides In between, each polypeptide was recognized using the method of the present invention. Therefore, the reason why the calibration curve for the polypeptides consisting of amino acids 60 to 121 of nociceptin and midkine could not be prepared was that the retention of these polypeptides on the column was insufficient under the measurement conditions used this time. In order to keep the column well, it was considered necessary to increase the proportion of the aqueous mobile phase from the measurement conditions used this time.

  The lower limit of quantification obtained this time is the result of using 100 μL of the sample injection amount, and the sensitivity can be increased 10 times by increasing the sample injection amount to 1 mL. Further, it is said that the MS / MS apparatus (API 365) used for the measurement this time has a sensitivity difference of 50 times or more as compared with the current latest MS / MS apparatus (API 5000). From the above, it is considered that further sensitivity enhancement, that is, quantification at several pM to fM, is possible by changing the sample injection amount and the apparatus, and sensitivity comparable to or exceeding the immunological technique can be obtained. it was thought.

Example 13 (Comparison of both systems in simultaneous determination of multiple samples)
<Sample preparation>
10 μL of each of the above 18 kinds of polypeptide stock solutions (100 μM) was added to 9.82 mL of the following solutions (A) to (D) to prepare a polypeptide mixed sample solution (100 nM).
(A) Water (B) Acetic acid-water (4:96, v / v)
(C) Acetic acid-water-acetonitrile (4:66:30, v / v / v)
(D) Acetic acid-water-acetonitrile (4:46:50, v / v / v)
Further, 100 μL of this polypeptide mixed sample solution was added to 900 μL of a solution having the same composition to prepare a polypeptide mixed sample solution (10 nM). Further, a 1 nM polypeptide mixed sample solution was prepared in the same manner.

<Measurement conditions>
Mobile phase A: Acetic acid-water mixture (volume ratio 4: 100)
Mobile phase B: Acetic acid-acetonitrile-methanol mixture (volume ratio 4:50:50)
Mobile phase C: acetic acid column: C ₃₀ reverse phase column (RPAQUEOUS-AR-3: inner diameter 2.0 mm, length 35 mm, particle diameter 3 μm)
Column temperature: 50 ° C
Flow rate: 0.2 mL / min
Gradient:

  100 μL of each polypeptide mixed sample solution was introduced into both systems.

<Result>
When a polypeptide mixed sample solution having an acetonitrile content of 30% or 50% was measured using a conventional method, all 18 types of polypeptides were hardly retained on the column regardless of the polypeptide concentration (C and D in FIG. 9). ). On the other hand, when a polypeptide mixed sample solution containing no acetonitrile was measured using the conventional method, all the polypeptides were retained on the column, but as the concentration decreased, a strongly retained polypeptide peak was observed at all. It disappeared (A and B in FIG. 9).
Next, when a polypeptide mixed sample solution not containing acetonitrile was measured using the method of the present invention, results almost similar to the conventional method were obtained (A and B in FIG. 10). However, when a polypeptide mixed sample solution having an acetonitrile content of 30% and 50%, which was hardly retained on the column by the conventional method, was measured by the method of the present invention, even a low concentration sample of 1 nM was retained. A strong polypeptide peak was observed, the size of which was almost proportional to the concentration (C and D in FIG. 10).

FIG. 11 shows the peak area of urocortin as an example in which isoleucyl-seryl-bradykinin is shown as an example in which almost no adsorption is observed and the retention is strong and the effect of adsorption is great.
The peak area of isoleucyl-seryl-bradykinin showed the same value in both the conventional method and the method of the present invention when a polypeptide mixed sample solution containing no acetonitrile was measured. On the other hand, when a polypeptide mixed sample solution having an acetonitrile content of 30% and 50% was measured, a decrease in peak area was observed in the conventional method, but when the method of the present invention was used, a polypeptide mixed sample not containing acetonitrile. A peak area similar to that obtained when the solution was measured was obtained. The reason why the peak area in the conventional method is reduced is that most of the isoleucyl-seryl-bradykinin introduced into the column without losing the adsorption ability to the column packing material due to acetonitrile exceeding the critical value contained in the sample solution This was thought to be due to passing through the column. Among the polypeptides examined this time, isoleucyl-seryl-bradykinin is a polypeptide that is relatively weakly retained in the column, and it is considered that adsorption to a container or the like at a low concentration is relatively small.
Next, the peak area of urocortin when a polypeptide mixed sample solution not containing acetonitrile was measured using a conventional method became extremely small as the concentration decreased. In particular, when a mixed sample prepared using only water was measured, no peak was observed except for 100 nM. The cause of this was thought that most of urocortin was lost due to adsorption to a container or the like during preparation of a dilution series and adsorption to a syringe during LC introduction. When the polypeptide mixed sample solution (100 nM) having an acetonitrile content of 30% and 50% was measured, the peak area was smaller than that measured when the polypeptide mixed sample solution not containing acetonitrile was measured. (1 nM and 10 nM) The peak of urocortin was also observed at the time of measurement, and the peak area was almost proportional to the concentration.
The peak area of urocortin when the polypeptide mixed sample solution not containing acetonitrile was measured using the method of the present invention was extremely small as the concentration decreased, as in the conventional method. However, when a polypeptide mixed sample solution having an acetonitrile content of 30% and 50% was measured, the peak area of urocortin was larger than the peak area obtained by the conventional method and was proportional to the concentration.
From the above results, it is possible to avoid the adsorption of the polypeptide at a low concentration by treating the polypeptide in the solution in a state where the adsorption ability to the solid is lost (for example, in a solution containing acetonitrile content higher than the critical value). It was shown that it is possible. Further, it was shown that a plurality of polypeptides can be simultaneously quantified by measuring the sample solution in this state using the method of the present invention.

Example 14 (CD spectrum analysis)
<Sample preparation>
0.11 mg of urocortin was dissolved in 4.0 mL of water-acetonitrile (volume ratio 90:10, 80:20, 70:30, 60:50 or 50:50) solution to obtain a 5.9 μM urocortin solution (E ) Was prepared.
On the other hand, 25 mg of isoleucyl-seryl-bradykinin was dissolved in 2.5 mL of water to prepare a 7.9 mM stock solution. 40 μL of this solution was added to 4.0 mL of water-acetonitrile (volume ratio 100: 0, 95: 5, 90:10, 85:15 or 80:20) to prepare a 79 μM solution.

<Measurement conditions>
To obtain a 250-200 nM CD spectrum of Urocortin and isoleucyl-seryl-bradykinin, the sample was transferred to a 10 mm square quartz cell (JASCO; Tokyo) and a J-720 type circular dichroism dispersometer (JASCO; Tokyo) Using. Each sample was measured 6 times (step resolution 1 nm, 1 search step), and an averaged spectrum was obtained.

<Result>
FIG. 12 shows CD spectra of urocortin solutions having different acetonitrile contents. The CD spectrum shows that the acetonitrile content in the solution increases from 10% to 40% at any measurement temperature, and the spectral intensity of 200-240 nM greatly decreases. In particular, from the decrease of the spectral intensity around 222 nM, Urocortin was thought to have a helical structure. On the other hand, even if the acetonitrile content in the solution increased from 40% to 50%, no significant change was observed in the CD spectrum. This change in the acetonitrile content of 40% or more was considered to correlate with the appearance of a peak having a retention time of 1.5 minutes observed in Example 1. Therefore, it was speculated that the phase transition of the adsorption ability of urocortin was caused by the change in the three-dimensional structure of urocortin by various organic solvents including acetonitrile.

  On the other hand, CD spectra of isoleucyl-seryl-bradykinin solutions having different acetonitrile contents are shown in FIG. Although it appears that there is no significant change overall, focusing on the spectrum around 220-240 nm, its spectral intensity decreased with increasing acetonitrile content in the solution. In particular, the decrease in the spectral intensity at 222 nm suggested that isoleucyl-seryl-bradykinin had a more helical structure with increasing acetonitrile content. At 40 ° C., even if the acetonitrile content was increased from 15% to 20%, no significant change was observed in the spectrum. The result at 40 ° C. was considered to be almost in agreement with the critical content of about 12% indicated by acetonitrile estimated from the retention time when the gradient gradient obtained in Example 7 was 1% / min. Therefore, it was speculated that the phase transition of the adsorption ability of isoleucyl-seryl-bradykinin was also caused by the conformational change caused by various organic solvents including acetonitrile.

  From these results, polypeptide elution in reverse-phase HPLC is controlled by the phase transition of the adsorption capacity of the polypeptide accompanying the change in the three-dimensional structure caused by the change in the organic solvent content in the eluent during gradient elution. it was thought.

Example 15 (Retention time of each polypeptide when using chromolis column and power law of gradient gradient)
<Sample preparation>
[Tyr (PO ₃ H ₂ ) ⁴ ] -angiotensin II and ovalbumin (323-339) stock solutions (each 1 mM), 10 μL each of 100 μM 20-type polypeptide stock solution, 10 μM and 10 mg / mL four-type polypeptide stock solution 10 μL each of a 10-fold diluted solution (100 μM) with water, and 10 μL of an angiotensin II stock solution (50 mM) diluted 50-fold with water and further diluted 10-fold with water (100 μM) in one Eppendorf tube Collected and added to 370 μL of water to prepare a mixed solution of 27 kinds of polypeptides so that the final concentration was 1 μM each (however, the concentration of ovalbumin was 1 mg / mL).

<Measurement conditions>
Mobile phase A: acetic acid-water (volume ratio 4: 100)
Mobile phase B: acetic acid-acetonitrile (volume ratio 4: 100), acetic acid-methanol (volume ratio 4: 100), acetic acid-ethanol (volume ratio 4: 100), acetic acid-isopropyl alcohol (volume ratio 4: 100), or 100% acetic acid column: _C18 reverse phase column (Chromolis Performance RP-18e: inner diameter 3.0 mm, length 100 mm)
Column temperature: 60 ° C
Flow rate: 0.2 mL / min
Gradient:

  10 μL of a 27-polypeptide mixed sample solution was introduced into the system.

<Result>
As a result of measuring the retention time of the polypeptide when the gradient gradient was changed to 0.5, 1, 2, 4, 6, 8, 10% / min by the conventional method (FIG. 1A), Regardless of the type of organic solvent used for the mobile phase, a good power law was observed between the retention time of the polypeptide retained on the column and the gradient gradient for all polypeptides of molecular weight 1007 to 45 kD used. (Table 44).

In this case, although the column temperature was 60 ° C., nociceptin and amyloid β-protein (1-16) were sufficiently retained in the column Chromolis Performance RP-18e used this time, and as a result, the retention time and the gradient gradient A power law showing a good correlation coefficient was observed between them. On the other hand, Nociceptin and amyloid β-protein (1-16) is the column temperature to _{C 4} column Develosil300C4-HG-5 used in Example 6 is hardly retained even if the 50 ° C., shows good correlation coefficient The power law could not be confirmed. The results of Example 8 (effect of column stationary phase) and Example 9 (effect of column temperature) and initial measurement conditions (100% mobile phase A = acetic acid-water (volume ratio 4: 100), flow rate 0. Considering the column pressure (about 2.1 MPa; 50 ° C.) exhibited by Develosil 300C4-HG-5 at 2 mL / min) and the column pressure (about 0.5 MPa; 60 ° C.) exhibited by Chromolis Performance RP-18e, The difference was not due to differences in column stationary phase and temperature, but was thought to be due to differences in column pressure during measurement. Therefore, taking into account the knowledge obtained so far, polypeptides exhibiting adsorption capacity for column packing materials at low column pressures have higher-order structures when subjected to column pressures above a certain level. It was thought that it could be changed to a higher-order structure (or a higher-order structure that does not exhibit adsorption ability) showing a smaller critical value. Therefore, a polypeptide that has been retained in a column under a low column pressure condition and has been eluted in an eluent (f = 1) that exhibits a critical value is less than or equal to the critical value (f <1) under a high column pressure condition. It was thought that it was eluted in the eluent showing).

  Chromolis Performance RP-18e is a silica rod type column (chromolis type column) in which the column skeleton and the flow path are integrated. Compared with a conventional particle packed column, it has a lower column back pressure under the same measurement conditions. This result confirmed the low column back pressure. In this way, the chromolis-type column is used for a polypeptide that can change to a higher-order structure (or a higher-order structure that does not exhibit adsorption capacity) showing a smaller critical value depending on the column pressure when a particle-packed column is used. Furthermore, it was considered that the low column back pressure can be sufficiently retained in the column, and it was considered effective in quantitative analysis of such a polypeptide.

  On the other hand, as in Example 6, using the retention time of each polypeptide obtained when the gradient gradient was 4% / min, 6% / min, and 8% / min, the entire measurement system was obtained from Equation (3). Table 45 shows the result of calculating the dead volume (average value ± SD). Regardless of the type of organic solvent used in mobile phase B and the gradient of the organic solvent, it was shown that the dead volume was almost constant and did not depend on the polypeptide. However, there was a tendency for the dead volume to become slightly smaller as the molecular weight of the polypeptide increased. This was thought to suggest that a small molecular weight polypeptide penetrated deeper into the column pores.

  Subsequently, Table 46 shows the retention time of each polypeptide obtained by measuring the gradient gradient at 1% / min. As described in Examples 6 and 7, the retention time of each polypeptide obtained by measuring the gradient gradient at 1% / min shows an approximate value of the organic solvent content that is the phase transition critical value of each polypeptide. It is thought that. However, since the aqueous mobile phase contains about 4% acetic acid at the start of measurement and the detected retention time contains about 4 minutes of dead volume, a polypeptide with a short retention time is used. Then, it is necessary to pay some attention to the handling of the obtained values.

  When the retention time obtained using this chromolis-type column was compared with the retention time obtained using the particle-packed column (Example 7), it was found that a polypeptide having a small retention, that is, a polypeptide having a small critical value, was dead. Although a relatively large difference that was considered to be caused by a difference in volume and a difference in column back pressure was observed, polypeptides that were sufficiently retained in the column such as urocortin showed almost the same value. Therefore, for a polypeptide having a short retention time, although some care is required in handling the obtained value, each polypeptide obtained by measuring the gradient gradient at 1% / min as described in Example 7 is used. It was considered that the retention time of the peptide showed an approximate value of the organic solvent content which is the phase transition critical value of each polypeptide.

Example 16 (Study of mouse plasma pretreatment method for determination of amyloid β-protein in plasma: deproteinization pretreatment method using organic solvent)
<Preparation of amyloid β-protein standard solution>
10 ml each of amyloid β-protein (1-38), amyloid β-protein (1-40), amyloid β-protein (1-42) and amyloid β-protein (1-43) stock solution (0.1 mM), The mixture was added to 960 μL of an acetic acid-water-acetonitrile mixture (volume ratio 4:50:50) to prepare an amyloid β-protein mixed solution (each 1 μM). Furthermore, 200 μL of this amyloid β-protein mixed solution (each 1 μM) was added to 300 μL of an acetic acid-water-acetonitrile mixed solution (volume ratio 4:50:50) to prepare an amyloid β-protein mixed solution (400 nM each). .

<Preparation of Amyloid β-protein-added mouse plasma>
10 μL of amyloid β-protein mixed solution (each 1 μM) was added to 490 μL of blank mouse plasma (Non-Sterile Mouse Plasma in Heparin, Rockland) to prepare amyloid β-protein-added mouse plasma (20 nM).

<Deproteinization method with methanol and acetonitrile>
Blank mouse plasma and 100 μL of amyloid β-protein-added mouse plasma (20 nM) were added to 400 μL of acetonitrile or methanol, and after stirring, centrifuged at 20,000 × g for 15 minutes (4 ° C.) to obtain a supernatant.

<Preparation of mouse plasma sample>
The supernatant obtained using amyloid β-protein-added mouse plasma (20 nM) was used as a mouse plasma sample.

<Preparation of reference sample>
10 μL of an amyloid β-protein mixed solution (400 nM each) was added to 990 μL of the supernatant obtained using blank mouse plasma to prepare reference samples (4 nM each).

<Measurement conditions>
Mobile phase A: acetic acid-water (volume ratio 4: 100)
Mobile phase B: 100% acetic acid mobile phase C: water-acetic acid-acetonitrile-methanol (volume ratio 20: 4: 40: 40)
Column: _C18 reverse phase column (Chromolis Performance RP-18e: inner diameter 3.0 mm, length 100 mm)
Column temperature: 60 ° C
Flow rate: 0.2 mL / min (however, 0.6 mL / min for 30 to 39.9 minutes)
Gradient:

  A mouse plasma sample and a 50 μL reference sample were introduced into the system.

<Recovery rate calculation method>
The recovery rate of each amyloid β-protein from mouse plasma was the ratio (%) of the peak area obtained when the mouse plasma sample was measured to the peak area obtained when the reference sample was measured.

<Result>
When amyloid β-protein-added mouse plasma is treated by a deproteinization pretreatment method using acetonitrile or methanol, which is generally used for analyzing low molecular weight compounds, mouse plasma of four types of amyloid β-protein is used. As shown in Table 48, the recovery rate from was low. The recovery rate was lower when acetonitrile was used than when methanol was used. In particular, the recovery rates of amyloid β-proteins (1-42) and (1-43) showed 0%. The reason for the low recovery rate of each amyloid β-protein is that each amyloid β-protein was incorporated into the aggregation process of the polymer polypeptide such as albumin during deproteinization using an organic solvent, or these polymers The co-precipitation of the polypeptide and each amyloid β-protein was considered as the cause. Therefore, it was considered that highly sensitive quantification of amyloid β-protein in mouse plasma using such an organic solvent deproteinization method was difficult.

Example 17 (Examination of mouse plasma pretreatment method for amyloid β-protein quantification: plasma dilution method with organic solvent containing acetic acid, effect of acetic acid content)
<Preparation of amyloid β-protein standard solution>
10 ml each of amyloid β-protein (1-38), amyloid β-protein (1-40), amyloid β-protein (1-42) and amyloid β-protein (1-43) stock solution (0.1 mM), The mixture was added to 960 μL of an acetic acid-water-acetonitrile mixture (volume ratio 4:50:50) to prepare an amyloid β-protein mixed solution (each 1 μM). Furthermore, 50 μL of this amyloid β-protein mixed solution (each 1 μM) was added to 450 μL of an acetic acid-water-acetonitrile mixed solution (volume ratio 4:50:50) to prepare an amyloid β-protein mixed solution (100 nM each). .

<Preparation of Amyloid β-protein-added mouse plasma>
10 μL of amyloid β-protein mixed solution (each 1 μM) was added to 490 μL of blank mouse plasma (Non-Sterile Mouse Plasma in Heparin, Rockland) to prepare an amyloid β-protein-added mouse plasma sample (20 nM).

<Plasma dilution method with organic solvent containing acetic acid>
950 μL of water-acetic acid-methanol (volume ratios 20:70:10, 20:60:20, 20:50:30, 20 with respect to 50 μL of water, blank mouse plasma or mouse plasma with amyloid β-protein (20 nM) : 40: 40 or 20:30:50) was added and stirred sufficiently. 400 μL of the mixed solution was added to a commercially available ultra-free MC centrifugal filter unit (Biomax-PB ultrafiltration membrane-equipped filter unit; molecular weight cut-off 10,000), and 6,000 × g for 60 minutes or more (35 ° C. ) Centrifuge to obtain a filtrate.

<Preparation of mouse plasma sample>
A mouse plasma sample was prepared by adding 300 μL of water-acetonitrile (volume ratio 20:80) to 300 μL of the filtrate obtained using amyloid β-protein-added mouse plasma (20 nM).

<Preparation of reference sample>
To 990 μL of the filtrate obtained using blank mouse plasma, 10 μL of an amyloid β-protein mixed solution (100 nM each) was added. 300 μL of water-acetonitrile (volume ratio 20:80) was added to 300 μL of this filtrate (1 nM each) to prepare reference samples (0.5 nM each).

<Preparation of control filtration sample>
10 μL of amyloid β-protein mixed solution (100 nM each) was added to 990 μL of the filtrate obtained using water instead of mouse plasma. A control filtration sample (each 0.5 nM) was prepared by adding 300 μL of water-acetonitrile (volume ratio 20:80) to 300 μL of this filtrate (1 nM each).

<Preparation of control sample>
After adding 50 μL of water to 950 μL of water-acetic acid-methanol (volume ratio 20:70:10, 20:60:20, 20:50:30, 20:40:40 or 20:30:50), amyloid 10 μL of β-protein mixed solution (100 nM each) was added. Furthermore, 300 μL of water-acetonitrile (volume ratio 20:80) was added to 300 μL of this solution (1 nM each) to prepare a control sample (0.5 nM each).

<Measurement conditions>
Mobile phase A: Water mobile phase B: Acetic acid-methanol-acetonitrile (volume ratio 10:45:45)
Mobile phase C: water-acetic acid-methanol-acetonitrile (volume ratio 40: 20: 20: 20)
Column: Non-porous C ₃₀ reverse phase column (NP C30-5: internal diameter 2.0 mm, length 100 mm, particle size 5 μm: custom-made column by Nomura Chemical)
Column temperature: 60 ° C
Flow rate: 0.2 mL / min
Gradient:

  Each control sample, control filtration sample, reference sample and mouse plasma sample 400 μL were introduced into the system.

<Recovery rate calculation method>
The recovery rate (permeability) of each amyloid β-protein when passing through the Biomax-PB ultrafiltration membrane used during the pretreatment operation is determined when the control filtration sample is measured with respect to the peak area obtained when the control sample is measured. The ratio (%) of the obtained peak area was used. On the other hand, the recovery rate of each amyloid β-protein from mouse plasma was the ratio (%) of the peak area obtained when the mouse plasma sample was measured to the peak area obtained when the reference sample was measured.

<Result>
From the results of Example 16, it was considered difficult to efficiently recover the target polypeptide from plasma while aggregating a high molecular polypeptide such as albumin using an organic solvent such as acetonitrile or methanol. . Therefore, a plasma dilution method combining acetic acid and an organic solvent was examined as a pretreatment method in which the high molecular polypeptide in plasma was not aggregated and the target polypeptide was turned into the OFF phase. This is because, in a solution containing a high content of acetic acid (generally about 20% or more depending on the dilution ratio with respect to plasma), even if an organic solvent is contained, the polypeptide in plasma is dissolved without aggregation. This method was devised based on the new knowledge that The plasma diluted with the mixed solution used in this study was all colorless and transparent, and it was considered that the plasma polypeptide was not aggregated. On the other hand, from the value f calculated from the acetic acid and methanol contents in a diluted mouse plasma sample (= filtrate) when the plasma is regarded as water, each amyloid β-protein is added to the column in the sample (= filtrate). It was suggested that the adsorption capacity for the filler was lost (Table 50). However, the retention time when the gradient gradient obtained in Example 15 was 1% / min was used as the phase transition critical value of each polypeptide for each organic solvent. Therefore, since each amyloid β-protein is considered to have lost the adsorption ability to containers that are in contact with the sample during preparation, the ability to adsorb to Biomax-PB ultrafiltration membranes has also been lost. Was expected.

  On the other hand, when using the method of the present invention, each mobile phase composition, the mixing ratio at the start of measurement of the organic solvent-based mobile phase (mixed solution of mobile phases B and C) into which the polypeptide is introduced, and each sample From the content of the organic solvent in the medium, the ratio (α) of the aqueous mobile phase to the organic mobile mobile phase mixed in the mixer required to hold each amyloid β-protein on the column is calculated according to the above-mentioned formula (a). (Table 51). However, the retention time when the gradient gradient obtained in Example 15 was 1% / min was used as the phase transition critical value of each polypeptide for each organic solvent. The ratio of the aqueous mobile phase necessary to retain all the amyloid β-protein introduced into the system on the column was calculated from α, and was found to be greater than 66%. In this study, the ratio of the aqueous mobile phase until the sample containing amyloid β-protein was introduced into the column was about 88%, and all the amyloid β-protein introduced into the system was retained in the column. It was judged.

<Result>
Actually, as a result of measuring each sample, the biomax-PB ultrafiltration membrane of each amyloid β-protein present in the diluted mouse plasma sample (= filtrate) exhibiting the OFF phase (f> 1) was obtained. The recovery was almost 100% ± 15% as expected (Table 52). Therefore, it was suggested that each amyloid β-protein has lost its adsorption ability to the Biomax-PB ultrafiltration membrane in a solution showing an OFF phase (f> 1).

  On the other hand, the recovery rate of each amyloid β-protein from mouse plasma increased with increasing acetic acid content in the solution used to dilute mouse plasma. In spite of the fact that each amyloid β-protein has lost its ability to adsorb substances in all solutions, one reason for the change in the recovery rate is that the molecular weight of each amyloid β-protein that is fractionated by the membrane is 10 It was thought that it was not recovered in the filtrate due to the strong interaction with more than 1,000 molecular polypeptides. Therefore, acetic acid was considered to have an effect of inhibiting the interaction between polypeptides in addition to the effect of phase transition of the ability of the polypeptide to adsorb to substances.

Example 18 (Examination of mouse plasma pretreatment method for quantification of amyloid β-protein: Effect of plasma dilution method / dilution ratio with organic solvent containing acetic acid)
<Preparation of amyloid β-protein standard solution>
10 ml each of amyloid β-protein (1-38), amyloid β-protein (1-40), amyloid β-protein (1-42) and amyloid β-protein (1-43) stock solution (0.1 mM), The mixture was added to 960 μL of an acetic acid-water-acetonitrile mixture (volume ratio 4:50:50) to prepare an amyloid β-protein mixed solution (each 1 μM). First, 50 μL of this amyloid β-protein mixed solution (each 1 μM) was added to 200 μL of an acetic acid-water-acetonitrile mixed solution (volume ratio 4:50:50) to prepare an amyloid β-protein mixed solution (200 nM each). . Next, 50 μL of an amyloid β-protein mixed solution (each 1 μM) was added to 450 μL of an acetic acid-water-acetonitrile mixed solution (volume ratio 4:50:50) to prepare an amyloid β-protein mixed solution (100 nM each). . In addition, 40 μL of an amyloid β-protein mixed solution (each 1 μM) was added to 960 μL of an acetic acid-water-acetonitrile mixed solution (volume ratio 4:50:50) to prepare an amyloid β-protein mixed solution (40 nM each). . Furthermore, 20 μL of an amyloid β-protein mixed solution (each 1 μM) was added to 980 μL of an acetic acid-water-acetonitrile mixed solution (volume ratio 4:50:50) to prepare an amyloid β-protein mixed solution (20 nM each).

<Preparation of Amyloid β-protein-added mouse plasma>
20 μL of amyloid β-protein mixed solution (each 1 μM) was added to 980 μL of blank mouse plasma (Non-Sterile Mouse Plasma in Heparin, Rockland) to prepare amyloid β-protein added mouse plasma (20 nM).

<Plasma dilution method with organic solvent containing acetic acid>
100-fold dilution: Blank mouse plasma and 10 μL of amyloid β-protein added mouse plasma (20 nM) were added to 990 μL of water-acetic acid-acetonitrile (volume ratio 19:40:40). 50-fold dilution: Blank mouse plasma and 20 μL of amyloid β-protein added mouse plasma (20 nM) were added to 980 μL of water-acetic acid-acetonitrile (volume ratio 18:40:40). 20-fold dilution: Blank mouse plasma and 50 μL of amyloid β-protein added mouse plasma (20 nM) were added to 950 μL of water-acetic acid-acetonitrile (volume ratio 15:40:40). 10-fold dilution: Blank mouse plasma and 100 μL of amyloid β-protein added mouse plasma (20 nM) were added to 900 μL of water-acetic acid-acetonitrile (volume ratio 10:40:40).
After sufficiently stirring the diluted mouse plasma, 400 μL thereof was added to a commercially available ultra-free MC centrifugal filter unit (Biomax-PB ultrafiltration membrane-equipped filter unit; molecular weight cut-off 10,000), and 6,000 Centrifugation was performed at xg for 60 minutes or more (35 ° C.) to obtain a filtrate. The number of filter units was increased as necessary.

<Preparation of mouse plasma sample>
Each filtrate obtained using amyloid β-protein-added mouse plasma (20 nM) was used as a mouse plasma sample (100-fold diluted mouse plasma sample, 50-fold diluted mouse plasma sample, 20-fold diluted mouse plasma sample, and 10-fold dilution). Mouse plasma sample).

<Preparation of reference sample>
100-fold diluted reference sample: prepared by adding 10 μL of amyloid β-protein mixed solution (20 nM each) to 990 μL of 100-fold diluted filtrate obtained using blank mouse plasma (each 0.2 nM). 50-fold diluted reference sample: Prepared by adding 10 μL of amyloid β-protein mixed solution (40 nM each) to 990 μL of 50-fold diluted filtrate obtained using blank mouse plasma (0.4 nM each). 20-fold diluted reference sample: Prepared by adding 10 μL of amyloid β-protein mixed solution (100 nM each) to 990 μL of 20-fold diluted filtrate obtained using blank mouse plasma (each 1 nM). 10-fold diluted reference sample: prepared by adding 10 μL of amyloid β-protein mixed solution (200 nM each) to 990 μL of 10-fold diluted filtrate obtained using blank mouse plasma (2 nM each).

<Measurement conditions>
Mobile phase A: acetic acid-water (4: 100; v / v)
Mobile phase B: 100% acetic acid mobile phase C: water-acetic acid-acetonitrile-methanol (20: 4: 40: 40; v / v / v / v)
Column: _C18 reverse phase column (Chromolis Performance RP-18e: inner diameter 3.0 mm, length 100 mm)
Column temperature: 60 ° C
Flow rate: 0.2 mL / min (however, 0.6 mL / min for 30 to 39.9 minutes)
Gradient:

  100-fold diluted mouse plasma sample and 100-fold diluted reference sample 500 μL, 50-fold diluted mouse plasma sample and 50-fold diluted reference sample 250 μL, 20-fold diluted mouse plasma sample and 100-fold diluted 20-fold diluted reference sample, and 10-fold diluted mouse plasma sample And 50 μL of a 10-fold diluted reference sample was introduced into the system.

<Recovery rate calculation method>
The recovery rate of each amyloid β-protein from mouse plasma was calculated as the ratio (%) of the peak area obtained when the mouse plasma sample was measured to the peak area obtained when the reference sample was measured.

  From the value f calculated from the acetic acid and acetonitrile content in the diluted mouse plasma sample (= filtrate) when the plasma is regarded as water, each amyloid β-protein is the column packing material in the sample (= filtrate). Further, it was considered that the adsorption ability was also lost for solids such as Biomax-PB ultrafiltration membranes and containers used for sample preparation (Table 54). However, the retention time when the gradient gradient obtained in Example 15 was 1% / min was used as the phase transition critical value of each polypeptide for each organic solvent.

  On the other hand, when using the method of the present invention, each mobile phase composition, the mixing ratio at the start of measurement of the organic solvent-based mobile phase (mixed solution of mobile phases B and C) into which the polypeptide is introduced, and each sample From the content of the organic solvent in the medium, the ratio (α) of the aqueous mobile phase to the organic mobile mobile phase mixed in the mixer required to hold each amyloid β-protein on the column is calculated according to the above-mentioned formula (a). (Table 55). However, the retention time when the gradient gradient obtained in Example 15 was 1% / min was used as the phase transition critical value of each polypeptide for each organic solvent. The ratio of the aqueous mobile phase required to retain all the amyloid β-protein introduced into the system from the column was calculated from α, and was found to be greater than 63%. In this study, the ratio of the aqueous mobile phase until the sample containing amyloid β-protein was introduced into the column was about 75%, and all the amyloid β-protein introduced into the system was retained in the column. It was judged.

<Result>
As a result of measuring each sample with different dilution ratios, the recovery rate of each amyloid β-protein from mouse plasma increased as the dilution ratio increased (Table 56). Although each amyloid β-protein has lost its ability to adsorb substances in all mouse plasma samples, the reason for this change in the recovery rate is that each amyloid β-protein is similar to the discussion in Example 17. It was considered that -protein was not recovered in the filtrate because it strongly interacted with a high molecular weight polypeptide having a molecular weight of 10,000 or more fractionated by the membrane. From this result, it was considered that the interaction between polypeptides is weakened by increasing the dilution ratio (decreasing the polypeptide concentration).

Example 19 (Study of mouse plasma pretreatment method for amyloid β-protein quantification: plasma dilution method with organic solvent containing acetic acid / effect of organic solvent type)
<Preparation of amyloid β-protein standard solution>
10 ml each of amyloid β-protein (1-38), amyloid β-protein (1-40), amyloid β-protein (1-42) and amyloid β-protein (1-43) stock solution (0.1 mM), The mixture was added to 960 μL of an acetic acid-water-acetonitrile mixture (volume ratio 4:50:50) to prepare an amyloid β-protein mixed solution (each 1 μM). 20 μL of this amyloid β-protein mixed solution (each 1 μM) was added to 980 μL of an acetic acid-water-acetonitrile mixed solution (volume ratio 4:50:50) to prepare an amyloid β-protein mixed solution (20 nM each).

<Preparation of Amyloid β-protein-added mouse plasma>
10 μL of amyloid β-protein mixed solution (each 1 μM) was added to 490 μL of blank mouse plasma (Non-Sterile Mouse Plasma in Heparin, Rockland) to prepare amyloid β-protein-added mouse plasma (20 nM).

<Plasma dilution method with organic solvent containing acetic acid>
Blank mouse plasma and 10 μL of amyloid β-protein added mouse plasma (20 nM) were added to 990 μL of the dilution mixture shown in Table 57. After sufficiently stirring the diluted plasma, 400 μL thereof was added to a commercially available ultra-free MC centrifugal filter unit (Biomax-PB ultrafiltration membrane-equipped filter unit; molecular weight cut-off 10,000), and 6,000 × Centrifugation was carried out at g for 60 minutes or more (35 ° C.) to obtain a filtrate. The number of filter units was increased as necessary.

<Preparation of mouse plasma sample>
A filtrate obtained using mouse plasma (20 nM) added with amyloid β-protein was used as a mouse plasma sample.

<Preparation of reference sample>
A reference sample (each 0.2 nM) was prepared by adding 10 μL of an amyloid β-protein mixed solution (20 nM each) to 990 μL of a 100-fold diluted filtrate obtained using blank mouse plasma.

<Measurement conditions>
Mobile phase A: acetic acid-water (4: 100; v / v)
Mobile phase B: Acetic acid mobile phase C: Water-acetic acid-methanol-acetonitrile mixture (volume ratio 20: 4: 40: 40)
Column: _C18 reverse phase column (Chromolis Performance RP-18e: inner diameter 2.1 mm, length 100 mm)
Column temperature: 60 ° C
Flow rate: 0.2 mL / min (however, 0.6 mL / min for 30 to 39.9 minutes)
Gradient:

  A mouse plasma sample and a 500 μL reference sample were introduced into the system.

<Recovery rate calculation method>
The recovery rate of each amyloid β-protein from mouse plasma was calculated as the ratio (%) of the peak area obtained when the mouse plasma sample was measured to the peak area obtained when the reference sample was measured.

  From the value f calculated from acetic acid and organic solvent content in a diluted mouse plasma sample (= filtrate) when the plasma is regarded as water, each amyloid β-protein is diluted mouse plasma sample (= filtrate). It was suggested that the adsorption capacity for the column packing material was lost. From the results of Example 17, at this time, each amyloid β-protein was considered to have lost its adsorption ability even for solids such as Biomax-PB ultrafiltration membranes and containers used for sample preparation (Table 59). ).

  On the other hand, when using the method of the present invention, each mobile phase composition, the mixing ratio at the start of measurement of the organic solvent-based mobile phase (mixed solution of mobile phases B and C) into which the polypeptide is introduced, and each sample From the organic solvent content, the ratio of the aqueous mobile phase to the organic mobile phase required to hold each amyloid β-protein on the column (β) and the amyloid in diluted mouse plasma samples with different organic solvent and acetic acid contents The ratio (γ) of the aqueous mobile phase to the organic mobile phase required to hold β-protein in the column was calculated based on the above-described formula (a) (Table 60 and Table 61). However, the retention time when the gradient gradient obtained in Example 15 was 1% / min was used as the phase transition critical value of each polypeptide for each organic solvent. Of the calculated β and γ, the higher value is α, and using this value, the ratio of the aqueous mobile phase necessary for each amyloid β-protein introduced into the system to be retained in the column was calculated. (Table 62). As a result, the ratio of the aqueous mobile phase for retaining the amyloid β-protein (1-38) in the water-acetic acid-isopropyl alcohol mixed solution (volume ratios 10:30:60 and 10:40:50) in the column It was shown to be greater than 75%, but for the other mixed solutions, it was shown to be 74% or less for the total amyloid β-protein. In this study, the ratio of the aqueous mobile phase until the sample containing amyloid β-protein is introduced into the column is about 75%, and when measuring the two kinds of solutions described above, amyloid β-protein Insufficient retention of (1-38) on the column was expected, but in other cases, all amyloid β-protein introduced into the system was considered retained on the column.

<Result>
Regardless of the type of organic solvent, the recovery of each amyloid β-protein from mouse plasma increased with increasing acetic acid content in the dilution solution (Table 65). On the other hand, when the acetic acid content in the diluted mixed solution was low (about 30% to 40%), the water content in the diluted mixed solution increased and the recovery rate tended to increase. However, when the acetic acid content was high (60% or more) and the water content was 20% or more, no significant difference was observed in the recovery rate. From this result, the acetic acid in the dilute mixed solution phase-adsorbed the polypeptide to the substance and also inhibited the interaction between the polypeptides, so that each amyloid β-protein mouse plasma The recovery rate was considered to have increased.

Example 20 (Evaluation of stability of amyloid β-protein in mouse plasma sample in autosampler)
<Preparation of amyloid β-protein standard solution>
10 ml each of amyloid β-protein (1-38), amyloid β-protein (1-40), amyloid β-protein (1-42) and amyloid β-protein (1-43) stock solution (0.1 mM), The mixture was added to 960 μL of an acetic acid-water-acetonitrile mixture (volume ratio 4:50:50) to prepare an amyloid β-protein mixed solution (each 1 μM).

<Preparation of Amyloid β-protein-added mouse plasma>
10 μL of amyloid β-protein mixed solution (each 1 μM) was added to 490 μL of blank mouse plasma (Non-Sterile Mouse Plasma in Heparin, Rockland) to prepare amyloid β-protein-added mouse plasma (20 nM). Blank mouse plasma 100 μL was added to 100 μL of this amyloid β-protein added mouse plasma (20 nM) to prepare amyloid β-protein added mouse plasma (10 nM). Similarly, 150 μL of blank mouse plasma was added to 150 μL of amyloid β-protein-added mouse plasma (10 nM), and amyloid β-protein-added mouse plasma (5 nM) was added to 100 μL of amyloid β-protein added mouse plasma (5 nM). Is added to mouse plasma (2.5 nM) supplemented with amyloid β-protein, 150 μL of blank mouse plasma is added to 150 μL of amyloid β-protein added mouse plasma (2.5 nM), and amyloid β-protein supplemented mouse plasma (1.25 nM) is added. 100 μL of blank mouse plasma was added to 100 μL of amyloid β-protein-added mouse plasma (1.25 nM), and amyloid β-protein-added mouse plasma was added. (0.625 nM) was prepared.

<Plasma dilution method using water-acetic acid-isopropyl alcohol mixed solution>
30 μL of amyloid β-protein-added mouse plasma was added to 1500 μL of water-acetic acid-isopropyl alcohol mixed solution (volume ratio 30:60:10). This water-acetic acid-isopropyl alcohol mixed solution (volume ratio 30:60:10) contains NPY (10 nM) as an internal standard (IS) polypeptide. After sufficiently stirring the diluted mouse plasma, 400 μL or less thereof is added to a commercially available ultra-free MC centrifugal filter unit (Biomax-PB ultrafiltration membrane-equipped filter unit; molecular weight cut-off 10,000), Centrifugation was performed at 000 × g for 60 minutes or more (35 ° C.) to obtain a filtrate. If necessary, the number of filter units was increased to obtain 1.2 mL or more of filtrate.

<Preparation of calibration curve sample>
The filtrate obtained using mouse plasma with amyloid β-protein (0.625, 1.25, 2.5, 5, 10, and 20 nM) was measured as a mouse plasma sample for a calibration curve (n = 1).

<Preparation of sample for stability evaluation in autosampler>
Filtrates obtained using amyloid β-protein-added mouse plasma (1.25 and 5 nM) were respectively prepared as samples for stability evaluation in an autosampler (n = 3).

<Measurement conditions>
Mobile phase A: acetic acid-water (4: 100; v / v)
Mobile phase B: Acetic acid mobile phase C: Water-acetic acid-methanol-acetonitrile mixture (volume ratio 20: 20: 30: 30)
Column: _C18 reverse phase column (Chromolis Performance RP-18e: inner diameter 2.1 mm, length 100 mm)
Column temperature: 60 ° C
Flow rate: 0.2 mL / min (however, 0.6 mL / min for 30.1-39.9 minutes)
Gradient:

  A 1000 μL mouse plasma sample was introduced into the system.

<Creation of calibration curve>
For the calibration curve, the peak area of each amyloid β-protein with respect to the peak area of NPY, which is IS, is plotted on the y-axis, the amyloid β-protein concentration (nM) is plotted on the x-axis, and 1 / (1 / x) of the concentration is weighted. It was created by the least square method used as. In creating a calibration curve, the ratio of the calibration curve sample concentration calculated using the created calibration curve (back-calculate) to the theoretical value is expressed as accuracy (%), and the accuracy is ± 15 except for the lower limit of quantification (LLOQ). The acceptable standard was within 20% and ± 20% at the lower limit of quantification (LLOQ). Furthermore, 75% or more of the calibration curve points used (including LLOQ and upper limit of quantification) satisfy this criterion.

<Stability in autosampler>
Samples for evaluating the stability in the autosampler (1.25 and 5 nM for each n = 3) were left in the autosampler set at 20 ° C. immediately after the preparation, and measurements were carried out at 28 hours and 37 hours after being left. Stability (%) was expressed as a ratio (accuracy) (%) of the concentration obtained from the calibration curve to the theoretical value.

<Result>
A peak area of each amyloid β-protein proportional to the concentration was obtained, and a calibration curve with a 40-fold concentration range having good accuracy within ± 15% of the theoretical value including LLOQ was obtained (Table). 65).

  Furthermore, as a result of measuring samples for stability evaluation in autosampler (1.25 and 5 nM), each amyloid β-protein existing in the filtrate after the pretreatment is stable in an autosampler at 20 ° C. for up to 37 hours. (± 66% of theoretical value) was shown (Table 66).

Example 21 (Stability evaluation of amyloid β-protein in mouse plasma)
<Preparation of amyloid β-protein standard solution>
10 ml each of amyloid β-protein (1-38), amyloid β-protein (1-40), amyloid β-protein (1-42) and amyloid β-protein (1-43) stock solution (0.1 mM), The mixture was added to 960 μL of an acetic acid-water-acetonitrile mixture (volume ratio 4:50:50) to prepare an amyloid β-protein mixed solution (each 1 μM).

<Preparation of Amyloid β-protein-added mouse plasma>
20 μL of amyloid β-protein mixed solution (each 1 μM) was added to 980 μL of blank mouse plasma (Non-Sterile Mouse Plasma in Heparin, Rockland) to prepare amyloid β-protein added mouse plasma (20 nM). Blank mouse plasma 100 μL was added to 100 μL of this amyloid β-protein added mouse plasma (20 nM) to prepare amyloid β-protein added mouse plasma (10 nM). Similarly, 100 μL of blank mouse plasma was added to 100 μL of amyloid β-protein-added mouse plasma (10 nM), and amyloid β-protein-added mouse plasma (5 nM) was added to 100 μL of amyloid β-protein added mouse plasma (5 nM). Is added to amyloid β-protein-added mouse plasma (2 nM), amyloid β-protein-added mouse plasma (2 nM) is added to 100 μL of blank mouse plasma, and amyloid β-protein-added mouse plasma (1 nM) is added to Add 100 μL of blank mouse plasma to 100 μL of added mouse plasma (1 nM) to prepare mouse plasma (0.5 nM) added with amyloid β-protein did. On the other hand, two samples of amyloid β-protein-added mouse plasma (15 nM) prepared by adding 150 μL of blank mouse plasma to 450 μL of amyloid β-protein-added mouse plasma (20 nM) were prepared as samples for stability evaluation.

<Plasma dilution method using water-acetic acid-isopropyl alcohol mixed solution>
30 μL of amyloid β-protein-added mouse plasma was added to 1500 μL of water-acetic acid-isopropyl alcohol mixed solution (volume ratio 30:60:10). This water-acetic acid-isopropyl alcohol mixed solution (volume ratio 30:60:10) contains NPY (10 nM) as an internal standard (IS) polypeptide. After thoroughly stirring the diluted mouse plasma, 400 μL or less of each solution is added to a commercially available ultra-free MC centrifugal filter unit (Biomax-PB ultrafiltration membrane-equipped filter unit; molecular weight cut-off 10,000). The filtrate was obtained by centrifugation at 3,000 × g for 60 minutes or more (35 ° C.). If necessary, the number of filter units was increased to obtain 1.2 mL or more of filtrate.

<Preparation of calibration curve sample>
The filtrate obtained using mouse plasma with amyloid β-protein (0.5, 1, 2, 5, 10 and 20 nM) was measured as a mouse plasma sample for a calibration curve (n = 1).

<Preparation of sample for stability evaluation in autosampler>
Mouse plasma for stability evaluation (15 nM) is left immediately after preparation, in ice-cooled (4 ° C.) and room temperature, and water-acetic acid-isopropyl acetate is prepared at 0.5 hours, 1 hour, 2 hours and 4 hours after preparation. A plasma stability evaluation sample was prepared using a plasma dilution method using an alcohol mixed solution (n = 1).

<Measurement conditions>
Mobile phase A: acetic acid-water (4: 100; v / v)
Mobile phase B: Acetic acid mobile phase C: Water-acetic acid-methanol-acetonitrile mixture (volume ratio 20: 20: 30: 30)
Column: _C18 reverse phase column (Chromolis Performance RP-18e: inner diameter 2.1 mm, length 100 mm) In-line column temperature: 60 ° C
Flow rate: 0.2 mL / min (however, 0.6 mL / min for 33.1 to 44.9 minutes)
Gradient:

  A 1000 μL mouse plasma sample was introduced into the system.

<Creation of calibration curve>
For the calibration curve, the peak area of each amyloid β-protein with respect to the peak area of NPY, which is IS, is plotted on the y-axis, the amyloid β-protein concentration (nM) is plotted on the x-axis, and 1 / (1 / x) of the concentration is weighted. It was created by the least square method used as. In creating a calibration curve, the ratio of the calibration curve sample concentration calculated using the created calibration curve (back-calculate) to the theoretical value is expressed as accuracy (%), and the accuracy is ± 15 except for the lower limit of quantification (LLOQ). The acceptable standard was within 20% and ± 20% at the lower limit of quantification (LLOQ). Furthermore, 75% or more of the calibration curve points used (including LLOQ and upper limit of quantification) satisfy this criterion.

<Plasma stability>
The stability of amyloid β-protein in mouse plasma was expressed as a ratio (accuracy) (%) of the concentration obtained from the calibration curve to the theoretical value.

<Result>
Under the measurement conditions used this time, since a contamination peak was observed at the elution position of amyloid β-protein (1-38), the other three types of amyloid β-protein were evaluated. As a result, a peak area of each amyloid β-protein that is proportional to the concentration is obtained, and a calibration curve with a 40-fold concentration range having good accuracy within ± 15% of the theoretical value including LLOQ is obtained. (Table 68).

  When a sample for stability evaluation in plasma (15 nM) was measured, the accuracy (%) of each amyloid β-protein concentration obtained when measuring a sample obtained from plasma left at room temperature was about 70 after 1 hour. About 4%, it decreased to about 26.7 to 50% after 4 hours. On the other hand, the accuracy (%) of each amyloid β-protein concentration obtained when measuring a sample obtained from plasma left under ice cooling (4 ° C.) is 98.2 to 105.0 after 4 hours. %, Suggesting that each amyloid β-protein is stable in plasma left under ice cooling (4 ° C.) (Table 69).

Example 22 (Accuracy and accuracy of quantification of amyloid β-protein in mouse plasma)
<Preparation of amyloid β-protein standard solution>
10 ml each of amyloid β-protein (1-38), amyloid β-protein (1-40), amyloid β-protein (1-42) and amyloid β-protein (1-43) stock solution (0.1 mM), The mixture was added to 960 μL of an acetic acid-water-acetonitrile mixture (volume ratio 4:50:50) to prepare an amyloid β-protein mixed solution (each 1 μM).

<Preparation of Amyloid β-protein-added mouse plasma>
10 μL of amyloid β-protein mixed solution (each 1 μM) was added to 490 μL of blank mouse plasma (Non-Sterile Mouse Plasma in Heparin, Rockland) to prepare amyloid β-protein-added mouse plasma (20 nM). 200 μL of blank mouse plasma was added to 200 μL of this amyloid β-protein added mouse plasma (20 nM) to prepare amyloid β-protein added mouse plasma (10 nM). Similarly, 200 μL of blank mouse plasma was added to 200 μL of amyloid β-protein added mouse plasma (10 nM), amyloid β-protein added mouse plasma (5 nM) was added to 200 μL of amyloid β-protein added mouse plasma (5 nM), and 300 μL of blank mouse plasma was added. Is added to amyloid β-protein-added mouse plasma (2 nM), amyloid β-protein-added mouse plasma (2 nM) is added to 200 μL of blank mouse plasma, and amyloid β-protein-added mouse plasma (1 nM) is added to 200 μL of blank mouse plasma is added to 200 μL of added mouse plasma (1 nM) to prepare mouse plasma (0.5 nM) added with amyloid β-protein. did. As a QC sample, 50 μL of blank mouse plasma was added to 150 μL of amyloid β-protein added mouse plasma (20 nM) to prepare amyloid β-protein added mouse plasma (15 nM).

<Plasma dilution method using water-acetic acid-isopropyl alcohol mixed solution>
30 μL of amyloid β-protein-added mouse plasma was added to 1500 μL of water-acetic acid-isopropyl alcohol mixed solution (volume ratio 30:60:10). This water-acetic acid-isopropyl alcohol mixed solution (volume ratio 30:60:10) contains NPY (10 nM) as an internal standard (IS) polypeptide. After thoroughly stirring the diluted mouse plasma, 400 μL or less of each solution is added to a commercially available ultra-free MC centrifugal filter unit (Biomax-PB ultrafiltration membrane-equipped filter unit; molecular weight cut-off 10,000). The filtrate was obtained by centrifugation at 3,000 × g for 60 minutes or more (35 ° C.). If necessary, the number of filter units was increased to obtain 1.2 mL or more of filtrate.

<Preparation of calibration curve sample>
The filtrate obtained using mouse plasma with amyloid β-protein (0.5, 1, 2, 5, 10 and 20 nM) was measured as a mouse plasma sample for a calibration curve (n = 1).

<Preparation of QC sample>
Filtrates obtained using amyloid β-protein added mouse plasma (0.5, 1, 5 and 15 nM) were prepared as LLOQ, LQC, MQC and HQC samples, respectively (n = 5).

<Measurement conditions>
Mobile phase A: acetic acid-water (4: 100; v / v)
Mobile phase B: Acetic acid mobile phase C: Water-acetic acid-methanol-acetonitrile mixture (volume ratio 20: 20: 30: 30)
Column: _C18 reverse phase column (Chromolis Performance RP-18e: inner diameter 2.1 mm, length 100 mm) In-line column temperature: 60 ° C
Flow rate: 0.2 mL / min (however, 0.25 mL / min for 0.1 to 10 minutes, 0.6 mL / min for 30 to 39.9 minutes)
Gradient:

  A 1000 μL mouse plasma sample was introduced into the system.

<Creation of calibration curve>
For the calibration curve, the peak area of each amyloid β-protein with respect to the peak area of NPY, which is IS, is plotted on the y-axis, and the amyloid β-protein concentration (nM) is plotted on the x-axis. It was created by the least square method used as. In creating a calibration curve, the ratio of the calibration curve sample concentration calculated using the created calibration curve (back-calculate) to the theoretical value is expressed as accuracy (%), and the accuracy is ± 15 except for the lower limit of quantification (LLOQ). The acceptable standard was within 20% and ± 20% at the lower limit of quantification (LLOQ). Furthermore, 75% or more of the calibration curve points used (including LLOQ and upper limit of quantification) satisfy this criterion.

<Calculation of accuracy and accuracy>
The accuracy (%) was expressed as a ratio (%) of the average concentration obtained when the QC sample (n = 5) was measured to the theoretical concentration, and the accuracy was expressed as a coefficient of variation (CV%). Acceptance criteria for accuracy (%) were within the theoretical value ± 15% except for the lower limit of quantification (LLOQ), and the theoretical value ± 20% at the lower limit of quantification (LLOQ). The tolerance criteria for accuracy were CV% within 15% except for the lower limit of quantification (LLOQ) and within 20% at the lower limit of quantification (LLOQ).

<Result>
Under the measurement conditions used this time, since a contamination peak was observed at the elution position of amyloid β-protein (1-38), the other three types of amyloid β-protein were evaluated. As a result, peak areas of amyloid β-protein (1-40), (1-42), and (1-43) proportional to the concentration were obtained, and good within ± 15% of the theoretical value including LLOQ A calibration curve with a 40-fold concentration range with high accuracy was obtained (Table 71). Furthermore, when the QC sample was measured, the accuracy and accuracy satisfying the acceptance criteria were obtained (Table 72).

  Since it was confirmed that amyloid β-protein (1-40), (1-42) and (1-43) in mouse plasma can be accurately quantified under the measurement conditions used this time, amyloid precursor protein ( Amyloid β-protein concentration in plasma of a transgenic mouse (Tg mouse) into which APP was recombinantly introduced was measured. The results are shown in Table 73. The concentration of amyloid β-protein (1-40) obtained by the method of the present invention was substantially correlated with the value obtained by the ELISA method separately. On the other hand, the concentration of amyloid β-protein (1-42) was not more than the lower limit of quantification of the peak detected. When the concentration obtained by extrapolating the calibration curve was used as a reference value and compared with the value obtained by the ELISA method, it was considered that there was almost a correlation. Therefore, it was shown that amyloid β-protein in biological samples such as plasma can be quantified by the method of the present invention.

  The pretreatment method and analysis method used this time are methods that can be applied to polypeptides other than amyloid β-protein, and further considering the power law to be recognized between the retention time and the gradient gradient, Then, in addition to being able to increase the sensitivity in proportion to the introduction of the sample amount as long as the sample introduction time and the column load allow, the MS / MS is about 50 times more sensitive than the API 365 used in this test. It is considered that further highly sensitive quantification is possible by using MS such as API5000. Therefore, it was considered that the method of the present invention enables a high-sensitivity quantification method for polypeptides in biological samples having sensitivity comparable to or surpassing immunological techniques by combining with the latest MS / MS. Further, as is clear from the present example, unlike the ELISA method, the method of the present invention can quantitate a plurality of polypeptides having different critical values at the same time, so that it is comprehensive as in current proteomics research. It was also considered effective in biomarker exploration research by polypeptide detection.

Claims (9)

A method for detecting or quantifying a polypeptide having a molecular weight of 1007 to 44 kDa (polypeptide A) using a reverse phase liquid chromatograph, comprising the following steps:
(1) introducing OFF-phase polypeptide A into a reversed-phase liquid chromatograph and moving it to a mixer with an organic solvent-based mobile phase;
(2) The mobile phase for polypeptide A phase transition is added to the OFF phase polypeptide A moved to the mixer in (1) by the mixer, and phase transition is made to the ON phase polypeptide A. Generating step,
(3) a step of allowing the ON phase polypeptide A produced in (2) to interact with the column filler,
(4) a step of causing the phase transition of the ON phase polypeptide A interacted in (3) to the OFF phase polypeptide A to produce the OFF phase polypeptide A,
(5) a step of eluting OFF-phase polypeptide A produced in (4), and (6) a step of detecting or quantifying polypeptide A eluted in (5),
Here, the OFF phase polypeptide is a polypeptide that does not substantially exhibit the adsorption ability to the reverse phase column filler, and the ON phase polypeptide is the polypeptide that exhibits the adsorption ability to the reverse phase column filler. OFF phase polypeptide A is one or more organic solvents selected from acetonitrile, methanol, ethanol, isopropyl alcohol, acetone, DMSO, THF, acetic acid, formic acid and TFA (organic solvent 1,..., Organic solvent n (n is an integer of 1 or more and 7 or less))), and is a polypeptide A existing in a solution (OFF phase solution) in which the following formula (1) is satisfied. A method for detecting or quantifying the polypeptide according to claim 1.
(In Formula (1), X1 is the phase transition critical value (%) of polypeptide A in the water-organic solvent 1 mixed solution, and Xn is the phase transition critical value (%) of polypeptide A in the water-organic solvent n mixed solution. X1 represents the volume ratio (%) of the organic solvent 1 in the OFF phase solution, and xn represents the volume ratio (%) of the organic solvent n in the OFF phase solution.) The method for detecting or quantifying a polypeptide according to claim 1, wherein the ON-phase polypeptide A is a polypeptide A present in a solution selected from the following group;
(1) an aqueous solution containing no organic solvent, and (2) one or more organic solvents selected from acetonitrile, methanol, ethanol, isopropyl alcohol, acetone, DMSO, THF, acetic acid, formic acid and TFA (organic solvent 1 A solution containing an organic solvent n (n is an integer of 1 or more and 7 or less), and satisfying the following formula (2) (ON phase solution).
(In the formula (2), X1 is the critical value of phase transition of polypeptide A in water-organic solvent 1 mixed solution (%), Xn is the critical value of phase transition of polypeptide A in water-organic solvent n mixed solution (%)) X1 represents the volume ratio (%) of the organic solvent 1 in the ON phase solution, and xn represents the volume ratio (%) of the organic solvent n in the ON phase solution) The polypeptide of claim 1, wherein the OFF phase polypeptide A is the polypeptide A represented by the following (A), and the ON phase polypeptide A is the polypeptide A represented by the following (B). Detection or quantification method;
(A) One or more organic solvents selected from acetonitrile, methanol, ethanol, isopropyl alcohol, acetone, DMSO, THF, acetic acid, formic acid and TFA (organic solvent 1,..., Organic solvent n (n is A polypeptide A present in a solution containing 1 or more and 7 or less))), wherein the polypeptide A is present in a solution (OFF phase solution) in which the following formula (1) is satisfied,
(In Formula (1), X1 is the phase transition critical value (%) of polypeptide A in the water-organic solvent 1 mixed solution, and Xn is the phase transition critical value (%) of polypeptide A in the water-organic solvent n mixed solution. , X1 represents the volume ratio (%) of the organic solvent 1 in the OFF phase solution, and xn represents the volume ratio (%) of the organic solvent n in the OFF phase solution) and (B) an aqueous solution containing no organic solvent, or acetonitrile , Methanol, ethanol, isopropyl alcohol, acetone, DMSO, THF, acetic acid, formic acid and TFA, or one or more organic solvents (organic solvent 1,..., Organic solvent n (n is 1 to 7) A polypeptide A present in a solution (ON phase solution) in which the following formula (2) is satisfied.
(In the formula (2), X1 is the critical value of phase transition of polypeptide A in water-organic solvent 1 mixed solution (%), Xn is the critical value of phase transition of polypeptide A in water-organic solvent n mixed solution (%)) X1 represents the volume ratio (%) of the organic solvent 1 in the ON phase solution, and xn represents the volume ratio (%) of the organic solvent n in the ON phase solution)   Determine the phase transition critical value of the adsorption ability of polypeptide A to the reversed-phase column packing material in each organic solvent and water mixed solution selected from the organic solvent contained in the OFF phase solution and the organic solvent contained in the ON phase solution. The method for detecting or quantifying the polypeptide according to claim 4, comprising a step of: The polypeptide detection or quantification method according to any one of claims 1 to 5, wherein the polypeptide A is any one polypeptide selected from the following group;
(1) a polypeptide comprising the first to 24th amino acid sequences of adrenocorticotropic hormone,
(2) a polypeptide comprising the first to the 16th amino acid sequence of β-amyloid,
(3) a polypeptide comprising the first to the 28th amino acid sequence of β-amyloid,
(4) a polypeptide comprising the first to the 38th amino acid sequences of β-amyloid,
(5) a polypeptide comprising the first to the 40th amino acid sequence of β-amyloid,
(6) a polypeptide comprising the first to the 42nd amino acid sequence of β-amyloid,
(7) A polypeptide consisting of the first to the 43rd amino acid sequence of β-amyloid,
(8) Growth hormone releasing factor,
(9) isoleucyl-seryl-bradykinin,
(10) insulin,
(11) Brain natriuretic peptide (BNP-32),
(12) C-type natriuretic peptide (CNP-53),
(13) a polypeptide comprising the amino acid sequence of positions 60 to 121 of midkine;
(14) Neuromedin C,
(15) Neuropeptide Y (NPY),
(16) nociceptin,
(17) oxytocin,
(18) urocortin,
(19) Midkine,
(20) interferon-γ,
(21) Atrial natriuretic peptide (ANP (1-28)),
(22) Rat neutrophil chemotactic factor-1 (CINC-1 / gro),
(23) Parathyroid hormone (PTH (1-84)),
(24) a polypeptide comprising the amino acid sequence of 323 to 339 amino acid sequence of ovalbumin,
(25) Ovalbumin,
(26) Angiotensin II, and (27) Angiotensin II in which the fourth tyrosine of the amino acid sequence is phosphorylated. The step of detecting or quantifying polypeptide A is a step of detecting or quantifying polypeptide A by means of polypeptide detection or quantification provided in a mass spectrometer connected to a reverse phase liquid chromatograph. 6. A method for detecting or quantifying the polypeptide according to any one of 6 above.   Mobile phase supply (mobile phase 1 supply) that supplies at least a mobile phase (mobile phase 1), mobile phase supply (mobile phase 2 supply) that supplies a mobile phase (mobile phase 2) different from mobile phase 1 ), A mobile phase supply device (mobile phase 3 supply device) for supplying a mobile phase (mobile phase 3) different from the mobile phases 1 and 2, a mobile phase 1 supply device and a mobile phase 2 supply device. A mobile phase mixer (mixer A) connected via a sample injector, a sample injector connected to the mixer A via a liquid feed tube, the mobile phase 3 supply device and the sample injector via a liquid feed tube A mobile phase mixer to be connected (mixer B), a reverse phase analysis column connected to the mixer B via a liquid feeding pipe, and a mass spectrometer connected to the reverse phase analysis column via a liquid feeding pipe; Liquid chromatograph / mass spectrometer (LC-MS) or liquid chromatograph / tandem mass spectrometer (LC-MS / M ).   Mobile phase supply (mobile phase 1 supply) that supplies at least a mobile phase (mobile phase 1), mobile phase supply (mobile phase 2 supply) that supplies a mobile phase (mobile phase 2) different from mobile phase 1 ), A mobile phase supply device (mobile phase 3 supply device) for supplying a mobile phase (mobile phase 3) different from the mobile phases 1 and 2, a mobile phase 1 supply device and a mobile phase 2 supply device. A mixer (mixer A) connected via a sample injector, a sample injector connected to the mixer A via a liquid feeding tube, and the mobile phase 3 supply device and the sample injector are connected via a liquid feeding tube. Mobile phase mixer (mixer B), reverse phase analysis column connected to mixer B via a liquid feed pipe, switching valve connected to the reverse phase analysis column via a liquid feed pipe, and the switching valve Liquid chromatograph / mass spectrometer (LC-MS) or liquid with connected mass spectrometer Chromatograph / tandem mass spectrometry (LC-MS / MS).