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US-12618849-B2 - Peptide analyzing method

US12618849B2US 12618849 B2US12618849 B2US 12618849B2US-12618849-B2

Abstract

In the analysis method according to the present invention, a predetermined peptide is separated by immunoprecipitation using an antibody which specifically binds to either an N-terminus or a C-terminus of the predetermined peptide. The separated predetermined peptide is digested with a protease to prepare peptide fragments, and among the peptide fragments, a peptide fragment at a terminus opposite to a terminus binding to the antibody is mass-spectrometrically detected.

Inventors

  • Ritsuko YODA

Assignees

  • SHIMADZU CORPORATION

Dates

Publication Date
20260505
Application Date
20191125

Claims (6)

  1. 1 . A peptide analyzing method comprising: separating Aβ1-42 from Aβ1-40 and APP669-711 by immunoprecipitation using an antibody which specifically binds to a C-terminus of Aβ1-42, or separating Aβ1-40 and APP669-711 from Aβ1-42 by immunoprecipitation using an antibody which specifically binds to a C-terminus of Aβ1-40 or APP669-711; preparing peptide fragments by digesting with a protease Aβ1-40 and APP669-711 separated; and mass-spectrometrically detecting, among the peptide fragments, a peptide fragment at the N-terminus of Aβ1-40 and APP669-711.
  2. 2 . The peptide analyzing method according to claim 1 , wherein the peptide fragments are detected by multiple reaction monitoring.
  3. 3 . The peptide analyzing method according to claim 1 , wherein the protease is trypsin.
  4. 4 . The peptide analyzing method according to claim 1 , wherein the protease is Lys-C.
  5. 5 . A method for analyzing peptides, comprising: preparing a biological sample containing plural types of peptides produced by cleaving a precursor protein at different cleavage sites with in vivo processing, wherein the plural types of peptides are generated from an amyloid precursor protein (APP); contacting the biological sample with an antibody that specifically binds to a C-terminus of Aβ1-42; separating, by immunoprecipitation, peptides that are bound to the antibody from those that are not bound to the antibody, among the plural types of peptides; digesting the separated peptides with an enzyme to obtain peptide fragments; analyzing the obtained peptide fragments by mass spectrometry; and based on the results of the mass spectrometry, making the following determinations: I) determining that an ion derived from a peptide fragment that has a sequence at an N-terminus corresponding to an N-terminus side of Aβ1-40 detected in the mass spectrometry is an ion derived from peptide fragment generated from Aβ1-40 present in the biological sample by the enzyme digestion, rather than an ion derived from peptide fragments generated from Aβ1-42 present in the biological sample by the enzyme digestion; and II) determining that an ion derived from peptide fragments that has a sequence at an N-terminus corresponding to an N-terminus side of APP669-711 detected in the mass spectrometry is an ion derived from peptide fragment generated from APP669-711 present in the biological sample, generated by the enzyme digestion.
  6. 6 . A method for analyzing peptides, comprising: preparing a biological sample containing plural types of peptides, wherein the plural types of peptides are generated from the amyloid precursor protein (APP) being cleaved at different sites due to in vivo processing; contacting the biological sample with an antibody that specifically binds to a C-terminus of Aβ1-40 or APP669-711; separating, by immunoprecipitation, peptides that are bound to the antibody from those that are not bound to the antibody, among the plural types of peptides; digesting the separated peptides with an enzyme to obtain peptide fragments; analyzing the obtained peptide fragments by mass spectrometry; and based on the results of the mass spectrometry, making the following determinations: I) determining that an ion derived from a peptide fragment that has a sequence at an N-terminus corresponding to an N-terminus side of Aβ1-40 detected in the mass spectrometry is an ion derived from a peptide fragment generated from Aβ1-40 present in the biological sample by the enzyme digestion, rather than an ion derived from peptide fragments generated from Aβ1-42 present in the biological sample by the enzyme digestion; and II) determining that an ion derived from peptide fragments that has a sequence at an N-terminus corresponding to an N-terminus side of APP669-711 detected in the mass spectrometry is an ion derived from a peptide fragment generated from APP669-711 present in the biological sample, generated by the enzyme digestion.

Description

CROSS REFERENCE TO RELATED APPLICATION This application relates to, but does not claim priority from, JP Ser. No. JP2017-096857 filed on May 15, 2017 and published as JP Pub. No. JP2018-194374 on Dec. 6, 2018, the entire contents of which are incorporated herein fully by reference. INCORPORATION BY REFERENCE OF SEQUENCE LISTING The content of the electronically submitted sequence listing, file name: Q250733_substitute sequence listing as filed.txt; size: 9,052 bytes; and date of creation: Dec. 30, 2025, filed herewith, is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to a peptide analyzing method. More specifically, the present invention relates to a method for mass-spectrometrically identifying and/or quantifying peptides cleaved at different positions by in vivo processing. Description of the Related Art Amyloid β (Aβ) is a peptide composed of about 40 amino acid residues and is thought to be deeply involved in development of Alzheimer's disease. It is known that an amyloid precursor protein composed of 770 amino acid residues (APP: SEQ ID NO: 1) is cleaved between Met671 and Asp672 by a β-secretase to cut out a C-terminus-side peptide (APP672-770), and this peptide is cleaved by a γ-secretase to produce Aβ. Aβ is attracting attention as a biomarker for Alzheimer's disease. Some trials to mass-spectrometrically quantify an amount of Aβ contained in a biological sample have been reported. For example, WO 2015/111430 reports a case that 22 types of APP-cleaved peptides were detected from a trace amount of human plasma by an analysis method of combining immunoprecipitation (IP) using anti-Aβ antibodies which specifically bind to Aβ (6E10 for which Phe4-Gly9 of Aβ is an epitope, and 4G8 for which Leu17-Val24 of Aβ is an epitope) with matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). WO 2015/111430 reports a case that a peptide cleaved at an N-terminus side of a site to be cleaved by β-secretase, such as APP669-711 (SEQ ID NO: 5), was detected as a peptide produced by APP processing. J. S. Kim et al., Analytica Chimica Acta, 2014, 840, 1-9 reports a case that a sample obtained by digesting all proteins in plasma with trypsin was analyzed by multidimensional LC/MS/MS to select Aβ17-28 as a surrogate peptide, and a total Aβ amount was quantified by multiple reaction monitoring (MRM; also referred to as selective reaction monitoring (SRM)). SUMMARY OF THE INVENTION In MALDI-MS, a concentration of a specific peptide in a sample can be quantified from a relative ratio of a peak intensity in a mass spectrum, but it cannot be said that MALDI-MS is suitable for quantifying an absolute content of a specific peptide. On the other hand, in MRM, a peptide is detected through MS/MS by selecting a combination of a specific precursor ion and product ion, and therefore MRM has an advantage that a trace amount of sample can be high-sensitively quantified with high selectivity from lots of impurities. In addition, a calibration curve method or an internal standard method enables quantitative analysis with higher accuracy than MALDI-MS. However, since an intermediate sequence of Aβ is selected as a surrogate peptide in the method described in J. S. Kim et al., Analytica Chimica Acta, 2014, 840, 1-9, Aβ having a different C-terminus cleavage site (e.g., Aβ1-38 (SEQ ID NO: 2), Aβ1-40 (SEQ ID NO: 3), Aβ1-42 (SEQ ID NO: 4), and an APP-cleaved peptide of which an N-terminus side is cleaved at a site other than the β-secretase cleavage site (between Met671 and Asp672 of APP) (e.g., the aforementioned APP669-711)) cannot be individually quantified. Examples of plural types of peptides produced by cleaving a precursor protein at different cleavage sites with in vivo processing include, besides Aβ produced from APP, a granulin produced from a progranulin, a parathyroid hormone (PTH) produced from a prepro-PTH, a brain natriuretic peptide (BNP) produced from a prepro-BNP, an adrenocorticotropic hormone (ACTH) produced from proopiomelanocortin, an α-melanocyte stimulating hormone (α-MSH), a β-MSH and a γ-MSH. For pathological diagnosis and the like, there is a need for an analytical technique capable of individually identifying/quantifying plural types of peptides produced by cleaving a precursor protein at different cleavage sites with in vivo processing. In the analysis method according to the present invention, a peptide is separated by immunoprecipitation (IP) using an antibody which specifically binds to either an N-terminus or C-terminus of a predetermined peptide such as an APP-cleaved peptide (e.g., Aβ). The peptide separated by IP is digested with a protease to prepare peptide fragments, and among the peptide fragments, a peptide fragment at a terminus opposite to a terminus binding to the antibody is mass-spectrometrically detected. The peptide fragments may also be mass-spectrometrically analyzed by multiple reaction moni