Disease detection method
Inventors
Kawakami, Daisuke • Matsubara, Toshiya • Uchida, Kazuhiko • Meno, Kohji
Assignees
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Publication Number
US-11592452-B2
Publication Date
2023-02-28
Expiration Date
Abstract
The present invention is a method for detecting a specific disease based on the result of a measurement in which the amount of a peptide serving as a biomarker contained in a biological sample is determined by using an LC-MS. A pretreatment process performed before the measurement using the LC-MS includes the steps of preparing a mixed sample solution by adding a stable isotope reagent and a trifluoroacetic acid to the biological sample, where the stable isotope reagent is prepared beforehand by labeling the peptide with a stable isotope; boiling the mixed sample solution; injecting the mixed sample solution after boiled into a solid-phase extraction column to make the peptide be retained in the solid-phase extraction column; and passing a water-soluble organic solvent through the solid-phase extraction column to elute the peptide retained in the solid-phase extraction column and collect the eluate.
Core Innovation
The invention is a disease detection method that detects a specific disease based on a measurement result in which an amount of a peptide serving as a biomarker contained in a biological sample is determined using a chromatograph mass spectrometer. The chromatograph mass spectrometer includes a liquid chromatograph unit and a mass spectrometer unit capable of multiple reaction monitoring (MRM) measurement. The method includes separating the sample solution for mass spectrometry in the liquid chromatograph unit and performing an MRM measurement under measurement conditions including a plurality of MRM transitions.
The method performs a pretreatment process before the measurement using the chromatograph mass spectrometer. The pretreatment process includes preparing a mixed sample solution by adding a stable isotope reagent and trifluoroacetic acid to the biological sample, where the stable isotope reagent is prepared beforehand by labeling the peptide with a stable isotope. The mixed sample solution is boiled, then injected into a solid-phase extraction column so that the peptide is retained in the column.
A water-soluble organic solvent is passed through the solid-phase extraction column to elute the peptide retained in the column and to collect the eluate. The water-soluble organic solvent comprises 50%-100% methanol or comprises 0.1%-1% trifluoroacetic acid and 10%-99.9% acetonitrile, and conditional downstream preparation is performed based on whether evaporation and/or dilution are required depending on the acetonitrile concentration.
Claims Coverage
The independent claim describes a complete disease detection method comprising a pretreatment workflow with stable isotope reagent and trifluoroacetic acid, solid-phase extraction and elution under defined solvent ranges, and an LC-MS/MS MRM measurement with multiple MRM transitions defined by precursor/product ion combinations. The independent claim contains multiple inventive features that jointly define the method.
Stable isotope labeling with trifluoroacetic acid pretreatment
Preparing a mixed sample solution by adding a stable isotope reagent and a trifluoroacetic acid to the biological sample, where the stable isotope reagent is prepared beforehand by labeling the peptide with a stable isotope.
Boiling and solid-phase extraction retention and elution
Boiling the mixed sample solution; injecting the mixed sample solution after boiled into a solid-phase extraction column to make the peptide be retained; and passing a water-soluble organic solvent through the solid-phase extraction column to elute the peptide retained and collect the eluate.
LC separation and MRM with precursor/product combinations by fragmentation locations
Separating the sample solution for mass spectrometry in the liquid chromatograph unit and performing an MRM measurement in the mass spectrometer unit under measurement conditions including a plurality of MRM transitions corresponding to combinations of a precursor ion and a plurality of kinds of product ions formed from the precursor ion by fragmentation at different locations in the precursor ion.
Defined water-soluble organic solvent composition and conditional downstream preparation
The water-soluble organic solvent comprises 50%-100% methanol or comprises 0.1%-1% trifluoroacetic acid and 10%-99.9% acetonitrile, with conditional downstream preparation based on the acetonitrile concentration.
Across the independent claim, the inventive concept is a disease detection method combining stable isotope reagent labeling with trifluoroacetic acid pretreatment, boiling and solid-phase extraction with defined water-soluble organic solvent elution, and LC separation followed by MRM using precursor/product ion transition combinations defined by fragmentation at different locations, with conditional downstream preparation steps based on the elution solvent composition and acetonitrile concentration.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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