Analysis method, peptide antioxidizing agent for use in MAKK spectrometry and kit for use in mass spectrometry

Patent metadata

Field	Value
Publication number	US-12498380-B2
Application number	US-201916965117-A
Country	US
Kind code	B2
Filing date	Jan 28, 2019
Priority date	Jan 29, 2018
Publication date	Dec 16, 2025
Grant date	Dec 16, 2025

Abstract

An analysis method includes: preparing a sample containing peptide to be analyzed and an antioxidizing agent for preventing oxidation of methionine, the antioxidizing agent containing a protein, ionizing the peptide by laser desorption/ionization; and mass-separating and detecting the peptide that has been ionized.

First claim

The invention claimed is:

1 . An analysis method for analyzing peptide containing methionine comprising:
preparing an analyzed sample containing a methionine-oxidized form and a methionine-non-oxidized form of a peptide to be analyzed, wherein the methionine non-oxidized form of the peptide has no oxidized sulfur atom in a side chain of oxide methionine residue; preparing a mixed sample containing the analyzed sample and an antioxidizing agent for preventing oxidation of a methionine residue of the methionine-non-oxidized form of the peptide on a sample plate for laser desorption/ionization mass spectrometry, the antioxidizing agent containing a protein; placing the mixed sample on the sample plate for laser desorption/ionization mass spectrometry; ionizing the mixed sample containing the peptide placed on the sample plate by laser desorption/ionization; and mass-separating the methionine-oxidized form and the methionine-non-oxidized form of the peptide contained in the mixed sample and detecting at least one of the methionine-oxidized form and the methionine-non-oxidized form of the peptide that has been ionized, wherein the protein contained in the antioxidizing agent prevents the oxidation of the methionine residue of the methionine-non-oxidized form of the peptide in the mixed sample by causing its own oxidation in the mixed sample.

2 . The analysis method according to claim 1 , wherein
the laser desorption/ionization is matrix-assisted laser desorption/ionization.

3 . The analysis method according to claim 1 , wherein
a concentration of the protein contained in the antioxidizing agent is 27.5 nM or more to less than 275 nM.

4 . The analysis method according to claim 3 , wherein
the concentration of the protein contained in the antioxidizing agent is 27.5 nM or more to less than 260 nM.

5 . The analysis method according to claim 4 , wherein
the concentration of the protein contained in the antioxidizing agent is 45.2 nM or more to less than 151 nM.

6 . The analysis method according to claim 1 , wherein:
the antioxidizing agent contains, besides the protein, an amino acid and/or reducing agent.

7 . The analysis method according to claim 6 , wherein
the amino acid and/or reducing agent is at least one compound selected from the group consisting of methionine, histidine, cysteine, tryptophan, dithiothreitol, tris (2-arboxyethyl) phosphine, 2-mercaptoethanol, tri-n-butylphosphine, dithioerythritol, ascorbic acid, polyphenol, sodium pyrosulfite, citric acid, glucose, carotene, tocopherol, thio-glycolic acid, N-acetylcysteine, hydroxylamine, reduced glutathione, 2-aminoethyl isothiouronium bromide, and thioglycerol.

8 . The analysis method according to claim 6 , wherein
a concentration of the amino acid and/or reducing agent is 0.001 mM or more to 10 mM or less.

9 . The analysis method according to claim 1 , further comprising:
purifying the peptide by affinity purification including eluting the peptide; wherein the protein is a ligand used in the affinity purification; and in the affinity purification, before or after elution of the peptide with an eluent, adding an amino acid and/or reducing agent to the eluent.

10 . The analysis method according to claim 9 , wherein
the amino acid and/or reducing agent is at least one compound selected from the group consisting of methionine, histidine, cysteine, tryptophan, dithiothreitol, tris (2-carboxyethyl) phosphine, 2-mercaptoethanol, tri-n-butylphosphine, dithioerythritol, ascorbic acid, polyphenol, sodium pyrosulfite, citric acid, glucose, carotene, tocopherol, thio-glycolic acid, N-acetylcysteine, hydroxylamine, reduced glutathione, 2-aminoethyl isothiouronium bromide, and thioglycerol.

11 . The analysis method according to claim 9 , wherein
a concentration of the amino acid and/or reducing agent is 0.0001 mM or more to 10 mM or less.

12 . The analysis method according to claim 1 , wherein
the sample plate includes a plurality of wells into which the mixed sample is place and an amount of the protein contained in the antioxidizing agent is 1 to 30 ng/well.

13 . The analysis method according to claim 12 , wherein
the amount of the protein contained in the antioxidizing agent is 3 to 30 ng/well.

14 . The analysis method according to claim 13 , wherein
the amount of the protein contained in the antioxidizing agent is 3 to 10 ng/well.

Assignees

• Shimadzu Corp

Inventors

• Kaneko Naoki

Classifications

• G01N2560/00

  Chemical aspects of mass spectrometric analysis of biological material · CPC title

• C07K1/22

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• H01J49/164

  Laser desorption/ionisation, e.g. matrix-assisted laser desorption/ionisation [MALDI] (sample holders H01J49/0418) · CPC title

• G01N27/628

  and a beam of energy, e.g. laser enhanced ionisation · CPC title

• G01N33/6851 Primary

  Methods of protein analysis involving laser desorption ionisation mass spectrometry · CPC title