Methods and systems for LC-MS/MS proteomic genotyping
Inventors
Shuford, Christopher Michael • Grant, Russell Philip • Bradley, Meghan Norris • Holland, Patricia Louise Miller • Levandoski, Michael
Assignees
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Publication Number
US-12163963-B2
Publication Date
2024-12-10
Expiration Date
Abstract
Disclosed are methods and systems using liquid chromatography/tandem mass spectrometry (LC-MS/MS and 2D-LC-MS/MS) for the proteomic analysis of genotypes. In certain embodiments, samples used in the analysis comprise dried bodily fluids.
Core Innovation
The disclosure provides a method and system for determining a genotype of a gene of interest in a subject using proteomic genotyping with dried bodily fluids. Blood is separated into blood cells and plasma on a solid substrate, and the plasma is allowed to dry on the solid substrate to generate a dried plasma sample containing a protein derived from the gene of interest.
The dried plasma sample is digested with trypsin in digestion buffer to generate at least one allele specific surrogate peptide for the protein. After digestion, mass spectrometry is used to detect the at least one allele specific surrogate peptide in the digested sample, and the genotype of the subject is determined based on the detecting results.
In one implementation, the mass spectrometry detection of allele specific surrogate peptides generates a proteomic profile of the allele specific surrogate peptides, and the genotype is determined based on the proteomic profile. The system includes a device for separating blood into red blood cells and plasma on a solid substrate, a station for digestion by trypsin in digestion buffer, an optional station for chromatographic purification, and a station for analyzing by mass spectrometry to detect the allele specific surrogate peptide and determine the genotype.
Claims Coverage
The partial content provides three independent claims: a method for determining genotype, a method using a dried plasma sample produced by separating plasma from red blood cells on the solid substrate prior to drying, and a system for determining genotype. Across these independent claims, the main inventive features are the solid-substrate drying of plasma, trypsin digestion to create allele specific surrogate peptides, and genotype determination from mass spectrometry detection of those peptides, including generation of a proteomic profile.
Solid-substrate separation and drying of plasma to form a dried plasma sample
Separating blood obtained from the subject into blood cells and plasma on a solid substrate and allowing the plasma to dry on the solid substrate, thereby generating a dried plasma sample containing a protein derived from the gene of interest.
Trypsin digestion to generate allele specific surrogate peptides
Digesting the dried plasma sample with trypsin in digestion buffer to generate at least one allele specific surrogate peptide for the protein.
Mass spectrometry detection and genotype determination from allele specific surrogate peptides
Using mass spectrometry to detect the at least one allele specific surrogate peptide in the digested sample and determining the genotype of the subject based on results of the detection.
Proteomic profile of allele specific surrogate peptides for genotype determination
Detecting the plurality of allele specific surrogate peptides in the digested sample to generate a proteomic profile and determining the genotype of the subject based on the proteomic profile.
System architecture with separation, digestion, optional purification, and mass spectrometry analysis
Providing a system comprising a device for separating blood into red blood cells and plasma on a solid substrate and allowing the plasma to dry, a station for digestion by trypsin in digestion buffer, an optional station for chromatographic purification of the allele specific surrogate peptide, and a station for analyzing by mass spectrometry to detect the allele specific surrogate peptide and determine the genotype.
The independent claims consistently tie genotype determination to dried plasma on a solid substrate, trypsin-generated allele specific surrogate peptides, and mass spectrometry detection of those peptides, with genotype determination based on peptide detection results or on a proteomic profile. The system claim further covers the device/station arrangement implementing these steps, including an optional chromatographic purification station and mass spectrometry analysis station.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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