Quantitation of GLA proteins by mass spectrometric analysis
Inventors
Prasad, Bhagwat • Thummel, Kenneth E. • Shaikh, Abdul Basit • Rettie, Allan E.
Assignees
University of Washington • Washington State University WSU
Publication Number
US-12105093-B2
Publication Date
2024-10-01
Expiration Date
2041-07-27
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Abstract
Methods of LC-MS/MS quantification of γ-carboxylated proteins in plasma, serum, or blood, including dried blood spots, are disclosed. The methods can be used to determine patient-specific dosing of anticoagulant drugs and diagnosis of liver diseases, such as hepatocellular carcinoma.
Core Innovation
The invention provides a novel method for quantifying γ-carboxylated proteins, γ-carboxylated protein proteoforms, and des-carboxylated proteins in biological samples, particularly those derived from vitamin K-dependent blood clotting factors such as prothrombin. The method involves extracting the relevant proteins from a small amount of biological sample, such as blood, plasma, or a dried blood spot, followed by proteolytic cleavage to generate γ-carboxylated and des-carboxylated peptides that can be analyzed.
Current methods, such as ELISA, are limited by their inability to distinguish or simultaneously quantify the different γ-carboxylated proteoforms of such proteins, and are not validated for many vitamin K-dependent proteins. As a result, there is a need for sensitive and accurate methods to quantify fully γ-carboxylated, partially γ-carboxylated, and des-carboxylated proteins arising from vitamin K-dependent blood clotting factors, which reflect the functional state and activity of these proteins in various clinical contexts.
The disclosed methods use liquid chromatography-mass spectrometry (LC-MS/MS), with optional derivatization of peptides to increase signal intensity, and employ multiple reaction monitoring to enable rapid, sensitive, and specific quantification of peptide profiles. This approach enables the determination of a patient-specific Gla-region peptide profile, which can be used for administration and titration of anticoagulant therapy and for diagnosis of blood clotting-related conditions including hepatocellular carcinoma and related diseases.
Claims Coverage
The patent contains one independent claim, which highlights the primary inventive features relating to a method for quantifying γ-carboxylated and des-carboxylated proteins in biological samples.
Extraction and enrichment of γ-carboxylated and des-carboxylated proteins from a biological sample
The method involves extracting proteins selected from γ-carboxylated proteins, γ-carboxylated protein proteoforms, and des-carboxylated proteins from a biological sample by creating a protein precipitate. The precipitate is enriched with the target proteins relative to the starting sample.
Separation and dissolution of enriched protein precipitate
The liquid extract (containing non-target components) is separated from the residual protein precipitate. The residual protein precipitate, now enriched with γ-carboxylated and des-carboxylated proteins, is dissolved in an aqueous medium to prepare it for further processing.
Proteolytic cleavage to generate peptide analytes
The dissolved, enriched protein solution is treated with a protease to produce a solution containing γ-carboxylated peptides, γ-carboxylated peptide proteoforms, and des-carboxylated peptides suitable for analysis.
Quantitative determination of peptide abundance
The method entails determining the quantity of the γ-carboxylated peptides, γ-carboxylated peptide proteoforms, and des-carboxylated peptides present in the resulting peptide solution.
Collectively, these inventive features establish a method that enables selective enrichment, cleavage, and quantification of specific γ-carboxylated and des-carboxylated protein forms from biological samples, enabling detailed protein modification profiling relevant for diagnostic and therapeutic purposes.
Stated Advantages
The invention requires only a small volume of biological sample, such as as little as 10 μL of blood, plasma, or a dried blood spot.
It provides rapid detection and quantification of fully γ-carboxylated peptides, partially carboxylated peptide proteoforms, and des-carboxylated peptides.
The method achieves increased mass spectrometry signal response and peptide detection through derivatization of the Gla amino acid side chain carboxyl moiety.
It enables sensitive and accurate quantification of proteins with variable post-translational γ-carboxylation modification, addressing limitations of existing assays such as ELISA.
The profile generated by the method can be used to guide anticoagulant therapy dosing and as a biomarker for diagnosing and managing disease states affecting clotting physiology.
Documented Applications
Determining the dose of anticoagulant therapies, such as warfarin, for a subject by generating a Gla-region peptide profile.
Diagnosis of hepatocellular carcinoma by comparing γ-carboxylated and des-carboxylated peptide profiles between subject samples and control samples.
Monitoring the impact of diseases such as Sars-CoV-2 (COVID-19) and cancer on clotting physiology by quantifying Gla proteins.
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