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US 10,705,093 B2Granted
Determination of small-molecule thiols and disulfides: protein bound cys and total cysteine as biomarkers of oxidative stress
AssigneeBloodworks
InventorsXiaoyun Fu, Shelby A. Cate, Jose Aron Lopez, Junmei Chen, Barbara A. Konkle
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Abstract
Compositions and methods for determining the level of thiol and disulfide containing molecules in a sample are provided. The compositions and methods can be used to determine the level of oxidative stress in a subject with or without antioxidant treatment. Also provided are biomarkers of oxidative stress.
Claims20
A method for measuring the total Cysteine (Cys) level in a sample,
- the method comprising:
- obtaining a sample comprising Cys containing molecules comprising free Cys and oxidized Cys;
- blocking free Cys oxidation in the sample by adding a blocking reagent to the sample to create a blocked Cys sample;
- adding an isotopically labeled Cys analogue to the blocked Cys sample to create a mixed Cys sample;
- reducing the oxidized Cys to free Cys by adding dithiothreitol (DTT) to the mixed Cys sample to create a reduced mixed Cys sample;
- blocking the free Cys within the reduced mixed Cys sample by adding the blocking reagent to the reduced mixed Cys sample to create a blocked mixed Cys sample;
- extracting blocked Cys from the blocked mixed Cys sample by adding methanol to the blocked mixed Cys sample to create a supernatant containing blocked Cys;
- and
- subjecting the supernatant containing blocked Cys to liquid chromatography-tandem mass spectrometry (LC-MS/MS) with Multiple Reaction Monitoring (MRM),
- thereby measuring the total Cys level in the sample.
7 more elements
A method for determining oxidative stress in a subject, the method comprising measuring the total Cysteine (Cys) level in a sample derived from a subject comprising:
- obtaining a sample comprising Cys containing molecules comprising free Cys and oxidized Cys;
- blocking free Cys oxidation in the sample by adding a blocking reagent to the sample to create a blocked Cys sample;
- adding an isotopically labeled Cys analogue to the blocked Cys sample to create a mixed Cys sample;
- reducing the oxidized Cys to free Cys by adding dithiothreitol (DTT) to the mixed Cys sample to create a reduced mixed Cys sample;
- blocking the free Cys within the reduced mixed Cys sample by adding the blocking reagent to the reduced mixed Cys sample to create a blocked mixed Cys sample;
- extracting blocked Cys from the blocked mixed Cys sample by adding methanol to the blocked mixed Cys sample to create a supernatant containing blocked Cys;
- and
- subjecting the supernatant containing blocked Cys to liquid chromatography-tandem mass spectrometry (LC-MS/MS) with Multiple Reaction Monitoring (MRM),
- wherein an increased total Cys level as compared to a control value of total Cys level is indicative of oxidative stress in the subject.
6 more elements
Dependencies are inferred from the claim text.