JP6870468B2 - Peptide analysis method - Google Patents

Description

The present invention relates to a method for analyzing peptides. More specifically, the present invention relates to a method for identifying and / or quantifying peptides that are cleaved at a plurality of different positions by processing in vivo by mass spectrometry.

Amyloid β (Aβ) is a peptide consisting of about 40 amino acid residues and is thought to be deeply involved in the development of Alzheimer's disease. Amyloid precursor protein (APP: SEQ ID NO: 1) consisting of 770 amino acid residues is cleaved between Met671 and Asp672 by β-secretase to excise the C-terminal peptide (APP672-770), and this peptide is γ-secretase. It is known that Aβ is produced by cleavage.

Aβ is attracting attention as a biomarker for Alzheimer's disease, and several attempts to quantify the amount of Aβ contained in a biological sample by mass spectrometry have been reported. For example, in Patent Document 1, immunoprecipitation (IP) using an anti-Aβ antibody that specifically binds to Aβ (6E10 having Ph4-Gly9 of Aβ as an epitope and 4G8 having Leu17-Val24 of Aβ as an epitope) is used. , Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) has been reported to detect 22 types of APP-cleaving peptides in trace amounts of human plasma. In Patent Document 1, as a peptide generated by APP of processing, APP669- 711 (SEQ ID NO: 5) or the like, it is reported that peptides truncated at the N-terminal side of the cleavage site β -secretase is detected There is.

In Non-Patent Document 1, a sample obtained by digesting all proteins in plasma with trypsin is analyzed by multidimensional LC / MS / MS, Aβ17-28 is selected as a surrogate peptide, and multiple reaction monitoring (MRM; selective reaction monitoring (SRM)) is performed. ), It has been reported that the total amount of Aβ was quantified.

WO2015 / 111430 Pamphlet

J. S. Kim et al., Analytica Chimica Acta, 2014, 840, 1-9.

Although MALDI-MS can quantify the concentration of a specific peptide in a sample from the relative ratio of peak intensities in the mass spectrum, it cannot be said that it is suitable for quantifying the absolute content of a specific peptide. On the other hand, since MRM selects a specific combination of precursor ion and product ion and detects it by MS / MS, it has an advantage that a small amount of sample can be quantified with high selectivity and high sensitivity from a large number of impurities. .. In addition, the calibration curve method and the internal standard method enable more accurate quantitative analysis than MALDI-MS.

However, in the method described in Non-Patent Document 1, since the intermediate sequence of Aβ is selected as the surrogate peptide, Aβ having a different C-terminal cleavage site (for example, Aβ1-38 (SEQ ID NO: 2), Aβ1-40 (for example) SEQ ID NO: 3), beta] 1-42 (SEQ ID NO: 4) and, N-terminal side of the cleaved APP cleavage peptides other than β-secretase cleavage site (between Met671 and Asp672 of APP) (e.g., the aforementioned APP669- 711) individual Cannot be quantified.

Examples of producing multiple types of peptides by cleaving precursor proteins at different cleavage sites by in vivo processing include granulins produced from progranulin and preproPTH, in addition to Aβ produced from APP. Parathyroid hormone (PTH) produced from, brain natriuretic peptide (BNP) produced from prepro BNP, adrenocorticotropic hormone (ACTH) produced from proopiomelanocortin, α-melanocyte stimulating hormone (α-MSH) ), Β-MSH, γ-MSH and the like.

In pathological diagnosis and the like, there is a demand for an analytical method capable of individually identifying and quantifying a plurality of types of peptides produced by cleavage at different cleavage sites by processing precursor protein in vivo.

In the analytical method of the present invention, peptides are separated by an immunoprecipitation method (IP) using an antibody that specifically binds to either the N-terminal or the C-terminal of a predetermined peptide such as an APP-cleaving peptide (for example, Aβ). .. The peptide separated by IP is digested with a protease to prepare a peptide fragment, and among the peptide fragments, the peptide fragment at the end opposite to the end that binds to the antibody is detected by mass spectrometry. Mass spectrometry of peptide fragments may be performed by multiple reaction monitoring (MRM).

In the method of the present invention, one of the N-terminal and the C-terminal of the peptide is identified by IP using an antibody, and the other is identified by mass spectrometry. Therefore, peptides having different cleavage sites are individually detected and quantified. Is possible.

It is a schematic diagram which shows the capture by an antibody of the APP cleavage type peptide, and the cleavage position by a protease.

In the analysis method of the present invention, either one of the N-terminal and the C-terminal of the peptide is identified by an antibody and separated by immunoprecipitation (IP), and the peptide separated by IP is protease-digested to produce a peptide fragment. After that, the peptide fragment containing the terminal opposite to the antibody discrimination site (epitope) is analyzed by mass spectrometry. In this method, one end of the peptide is identified by the antibody and the other end of the peptide is identified by mass spectrometry. Therefore, a plurality of types of peptides produced by cleaving at multiple points in the living body are separated by amino acid sequence. Can be detected and quantified.

The peptide to be analyzed is not particularly limited, but the method of the present invention is suitable for analysis of a plurality of types of peptides produced by being cleaved at different sites by processing in vivo. Examples of precursor proteins that produce a plurality of types of peptides include amyloid precursor protein (APP), progranulin, prepro PTH, prepro BNP, and proopiomelanocortin. In the following, the analysis of peptides produced by the processing of APP (APP cleavage type peptides) will be mainly described.

[Separation of peptides by immunoprecipitation]
First, a predetermined peptide is separated and recovered from a biological sample by an immunoprecipitation method. Biological samples include blood samples, cerebrospinal fluid (CSF) urine, cerebrospinal fluid, saliva, body fluids such as sputum, feces and the like. Blood samples include whole blood, plasma, serum and the like. The blood sample may be prepared by appropriately treating whole blood collected from a living body. For example, when an APP-cleaving peptide is the target, a biological sample for screening for Alzheimer's disease or other diseases is the target sample for analysis.

By contacting the biological sample with the antibody, a predetermined peptide in the biological sample specifically binds to the antibody. As the antibody, an antibody that recognizes the peptide to be detected and specifically binds to the peptide is used. Examples of the antibody having an antigen-binding site that can recognize the APP-cleaving peptide include various anti-amyloid β antibodies.

In the present invention, an antibody having an amino acid sequence on the N-terminal side or C-terminal side of the APP-cleaving peptide as an epitope is used. The antibody may be monoclonal or polyclonal. The antibody may contain an antigen-binding site, and may be _{an antibody fragment (domain) such as F (ab') 2} , F (ab'), F (ab), Fd, or Fv. Due to its high binding specificity, antibody fragments that do not contain Fc domains such as F (ab'), F (ab), and Fv are preferable.

Antibodies with the N-terminal side of Aβ as an epitope include 3D6 (epitope: Aβ1-5), pAb-EL16 (epitope: Aβ1-7), 2H4 (epitope: Aβ1-8), 1E11 (epitope: Aβ1-8). , 20.1 (Epitope: Aβ1-10), pAb1-42 (Epitope: Aβ1-11), NAB228 (Epitope: Aβ1-11), Rabbit anti-Aβ polyclonal antibody (Abcam) (Epitope: Aβ1-14), AB10 (Epitope: Aβ1-16), 82E1 (Epitope: Aβ1-16), DE2 (Epitope: Aβ1-16), DE2B4 (Epitope: Aβ1-17), Rabbit anti-human Aβ polyclonal antibody (ABR) (Epitope: N-terminal of Aβ), etc. It has been known.

Antibodies having the C-terminal side of Aβ as an epitope include G2-10 (epitope: Aβ31-40), 1A10 (epitope: Aβ35-40), EP1876Y (epitope: Aβx-40), and G2-11 (epitope: Aβ33-). 42), 16C11 (Epitope: Aβ33-42), 21F12 (Epitope: Aβ34-42), D-17 goat anti-Aβ antibody (Epitope: C-terminal of Aβ1-42), BC05 (Epitope: C-terminal of Aβ1-42) Etc. are known.

For the purpose of improving the collection efficiency of peptides, these antibodies may be used in combination of two or more. Further, an antibody having an intermediate sequence of Aβ as an epitope may be used in combination. Antibodies using the intermediate sequence of Aβ as an epitope include 10D5 (epitope: Aβ3-7), 6E10 (epitope: Aβ4-9), WO-2 (epitope: Aβ4-10), 1A3 (epitope: Aβ5-9), pAb-EL21 (Epitope: Aβ5-11), 310-0 (Epitope: Aβ5-16), chicken anti-human Aβ polyclonal antibody (Abcam) (Epitope: Aβ12-28 or Aβ25-35), 12C3 (Epitope: Aβ10-16) ), 16C9 (Epitope: Aβ10-16), 19B8 (Epitope: Aβ9-10), pAb-EL26 (Epitope: Aβ11-26), BAM90.1 (Epitope: Aβ13-28), Rabbit anti-Aβ polyclonal antibody (MBL) (Epitope: Aβ15-30), 22D12 (Epitope: Aβ18-21), 266 (Epitope: Aβ16-24), pAb-EL17 (Epitope: Aβ15-24), 4G8 (Epitope: Aβ17-24), Rabbit anti-Aβ polyclonal Antibodies (Abcam) (epitope: Aβ22-35) and the like are known.

The antibody may be immobilized on any carrier. Carriers used for antibody immobilization include agarose, sepharose, dextran, silica gel, polyacrylamide, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, (meth) acrylic acid-based polymers, fluororesins, metal complex resins, and glass. Examples include metal. The antibody may be bound to the carrier via a spacer.

IP using the above antibody can be carried out by a known method. For example, when an antibody immobilized on a carrier is used, the target peptide may be bound to the antibody on the surface of the carrier, the components not bound to the antibody may be washed and removed, and then the peptide may be dissociated from the antibody to collect the eluate. Good.

The biological sample may be pretreated before binding the antibody to the peptide in the biological sample. For example, by contacting a biological sample with a carrier to which protein G, protein A, etc. are bound to remove antibodies such as IgG and IgM contained in blood, etc., non-specific adsorption with peptides to be analyzed such as Aβ, etc. Can be suppressed, the specificity of binding between the above-mentioned Aβ-specific binding antibody and the peptide to be analyzed can be enhanced, and the quantitativeness of analysis can be improved.

As a solution for binding the antibody and the peptide, a buffer solution having a pH of about 6.5 to 8.5 containing a surfactant is preferably used in order to suppress non-specific adsorption. As the surfactant, those that are less likely to cause denaturation of proteins such as antibodies and are easily removed by washing are preferable, and a neutral surfactant having maltose in the hydrophilic part and a neutral surfactant having trehalose in the hydrophilic part are preferable. Examples thereof include an activator and a neutral surfactant having glucose in the hydrophilic portion. Examples of the buffer solution include Tris buffer solution, phosphate buffer solution, HEPES buffer solution and the like.

After binding the antibody and the peptide, the contaminants that are not bound to the antibody are removed. When an antibody immobilized on a carrier is used, impurities may be removed by washing with a washing solution. For example, it is preferable to perform cleaning with a neutral buffer solution containing a surfactant, and then perform cleaning with an aqueous solution containing ammonium ions.

After washing, the peptide is separated and recovered by dissociating the peptide from the antibody. For example, by contacting the surface of the carrier on which the antibody is immobilized with the eluate, the peptide is dissociated from the antibody, and the dissociated peptide is eluted. As the eluate, an acidic aqueous solution having a pH of about 1 to 4 is generally used. The eluate may contain an organic solvent such as acetonitrile, acetone, methanol, ethanol, isopropanol, and chloroform.

[Protease treatment and mass spectrometry]
Peptides separated by immunoprecipitation are fragmented by protease digestion. Generally, a peptide denaturation treatment and an alkylation treatment are performed prior to the protease treatment. The conditions for the modification treatment and the alkylation treatment are not particularly limited, and known conditions are appropriately adopted.

The conditions for protease treatment are not particularly limited, and an appropriate protocol according to the protease used is adopted. For example, it is preferable to incubate for about 4 to 20 hours at a temperature of about 37 ° C. in a buffer solution adjusted to the optimum pH of the protease.

Proteases recognize amino acid sequences and selectively cleave specific bonds of specific sequences. The proteases include trypsin (cleaved position: C-terminal side of basic amino acid residues (Arg and Lys)), Lys-C (cleaved position: C-terminal side of Lys), and arginine endopeptidase (cleaved position: C-terminal side of Arg). Side), chymotrypsin (cleaved position: C-terminal side of aromatic amino acids (Phe, Tyr and Trp)), pepsin (cleaved position: N-terminal side of aromatic (Phe, Tyr and Trp)), Asn-C (cleaved position) : C-terminal side of Asn) and the like are used.

The protease may be selected so that a fragment having an amino acid sequence specific to the peptide to be analyzed can be obtained. Proteases may be used in combination of two or more. When the peptide fragment is detected by MRM, it is preferable to use a peptide fragment consisting of 6 or more amino acid residues as a precursor ion to be detected from the viewpoint of ensuring specificity. The upper limit of the length of the peptide fragment is not particularly limited, but the number of amino acid residues is preferably 30 or less because ionization is easy.

FIG. 1 is a schematic view showing the capture of a peptide by immunoprecipitation using an antibody that specifically binds to the C-terminal side of three types of APP-cleaving peptides, and the cleavage position of the peptide by a protease. The cleavage positions I to IV in the figure are the cleavage positions by trypsin (the C-terminal side of Lys residue and Arg residue). The cleavage position II shown by the broken line in the figure is on the C-terminal side of the Arg residue, and is cleaved by trypsin but not by Lys-C.

When IP is performed with the antibody a having the C-terminal of Aβ1-42 as an epitope, only Aβ1-42 having Ala42 at the C-terminal is captured among the three peptides shown in FIG. After digesting the peptide captured by IP using antibody a with trypsin, the presence of Aβ1-42 was confirmed by detecting the peptide fragment having the N-terminal sequence "DAEFR" of cleavage site II by mass spectrometry. it can. Trypsin digestion of Aβ1-40 also produces a peptide fragment having the sequence "DAEFR", but since Aβ40 is not captured by IP using antibody a, only the peptide fragment derived from Aβ1-42 is detected.

When a peptide captured by IP using antibody a is digested by Lys-C, if a peptide fragment having the N-terminal sequence "DAEFRHDSGYEVHHQK" of cleavage site III is detected by mass spectrometry, Aβ1-42 The existence can be confirmed. When trypsin was used, the number of amino acid residues in the peptide fragment on the N-terminal side generated from Aβ1-42 was as small as 5, whereas when Lys-C was used, the peptide fragment with 16 amino acid residues was present. Since it is obtained, it is suitable for detection and quantification of peptide fragments by MRM.

When performing the IP by antibody b to epitope C-terminus of [beta] 1-40, in the three peptides shown in FIG. 1, the two [beta] 1-40 and APP 669 -711 is captured. Captures Aβ1-42 by IP using antibody a, the outflow solution without being trapped in the antibody a (including wash) was conducted IP using an antibody b, and Aβ1-40 and APP 669 -711 You may capture it.

From digested sample by IP captured peptide by Lys-C using the antibody b, peptides having the sequence "DAEFRHDSGYEVHHQK" N-terminal side of the cutting position III of the [beta] 1-40, and the cutting position of the APP 669 -711 A peptide having the N-terminal sequence of III (sequence between cleavage positions I and III) " M DAEFRHDSGYEVHHQK" is produced. Since these peptides have different molecular weights, they can be identified by mass spectrometry.

IP alone with antibodies b, but the sequence of the C-terminal can not identify the to Aβ1-40 and APP 669 -711 common to separate the peptides by IP to the C-terminal side and the antibody recognition site, captured by IP By selecting the N-terminal protease digestion fragment of the recovered peptide as the transition, the difference in the N-terminal sequence (difference in the cleavage site from APP in vivo) can be identified.

In the present invention, after separating the peptide by IP using an antibody that specifically binds to either the C-terminal or the N-terminal of the peptide as described above, the peptide fragment at the terminal opposite to the antibody binding site is used. The analysis is performed. For example, in the peptide separated by IP using an antibody that binds to the C-terminal side of Aβ, the protease digested fragment on the N-terminal side is selected as a detection target by mass spectrometry after protease treatment. Peptides captured by IP using an antibody that binds to the N-terminal side of Aβ are subjected to mass spectrometry of the protease digested fragment on the C-terminal side. Three peptides shown in Figure 1 is IP by separation and beta] 1-42 and Aβ1-40 and APP 669 -711 C-terminal side, by mass spectrometry of the peptide fragments of the N-terminal side, beta] 1-42 and Aβ1- It can be distinguished from 40.

In the protease digestion performed after IP, it is preferable to select the protease so that the number of amino acid residues of the peptide fragment to be detected is within the above range. When the C-terminal side of Aβ is identified by IP and the N-terminal side is identified by mass analysis, the number of amino acid residues in the trypsin digested fragment on the N-terminal side is 5, and the number of amino acid residues in the Lys-C digested fragment. Is 16. It can be said that Lys-C is suitable as a protease in order to obtain an N-terminal peptide fragment having a length suitable for analysis by MRM.

The sample after the protease treatment may be subjected to treatments such as desalting, solubilization, concentration and drying, if necessary. For example, desalting and concentration may be performed using a spin column for solid-phase extraction or the like.

An apparatus is used for mass spectrometry to analyze peptide fragments obtained by protease treatment. Peptide fragment analysis can be performed by various mass spectrometric methods, but analysis by multiple reaction monitoring (MRM) is preferred due to its high selectivity and sensitivity. In MRM, separation by liquid chromatography (LC) is performed before the sample is introduced into the mass spectrometer.

The sample introduced into the mass spectrometer is ionized by an ionization probe. By mass spectrometry in the previous stage, ions (precursor ions) derived from the peptide fragment to be detected are selected. The precursor ion is cleaved into a plurality of types of ions (product ions) by CID or the like, and a specific product ion is separated based on m / z by mass spectrometry in the subsequent stage and detected by a detector. In MRM, a combination of m / z of precursor ions generated from the peptide to be analyzed and m / z of product ions selected by mass spectrometry in the subsequent stage is predetermined for each precursor ion.

As an example, the MRM transition of a peptide having the N-terminal sequence "DAEFRHDSGYEVHHQK" (SEQ ID NO: 6) of the Lys-C digest of Aβ1-42 (as well as the Lys-C digest of Aβ1-40) is shown below. ..

Based on the amount of product ions detected by MRM, the content (concentration) of a predetermined peptide in a biological sample such as blood can be calculated. The amount of product ions and the peptide concentration in the biological sample are associated with each other by predetermined parameters in advance. As a method of associating the two, for example, a method of using a calibration curve (calibration curve) according to an external standard can be mentioned. For the calibration curve, analyze the peptide fragment (external standard) with a known concentration under the same conditions as this analysis (analysis of biological sample), and plot the concentration and the peak area (or peak intensity) of the product ion. Obtained by

Claims (3)

Peptide analysis method
The predetermined peptide is separated by an immunoprecipitation method using an antibody that specifically binds to either the N-terminal or the C-terminal of the predetermined peptide.
A peptide fragment is prepared by digesting the separated predetermined peptide with a protease.
Among the peptide fragments, the peptide fragment at the end opposite to the end that binds to the antibody was detected by mass spectrometry.
The peptide is at least one of Aβ1-40 (SEQ ID NO: 3), Aβ1-42 (SEQ ID NO: 4) and APP669-711 (SEQ ID NO: 5).
Aβ1-42 and Aβ1-40 and APP669-711 have different C-terminal sequences, and Aβ1-40 and Aβ1-42 and APP669-711 have different N-terminal sequences. A method for analyzing a peptide, in which the precipitation method is performed at least twice. The method for analyzing a peptide according to claim 1, wherein the peptide fragment is detected by multiple reaction monitoring. The method for analyzing a peptide according to claim 1 or 2, wherein the predetermined peptide is a peptide produced by cleavage of amyloid precursor protein.

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