Methods for determining the redox status of proteins
AssigneeThe University of Western Australia Proteomics International Pty Ltd.
InventorsRichard J. Lipscombe, Peter G. Arthur, James K. Lui
Life of this patent
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Abstract
A method for determining the redox status of a protein sample, the method comprising the steps of: a) contacting the sample with a first label adapted to bind to at least one reduced cysteine group therein; b) contacting the sample with a reducing agent to reduce at least one oxidized cysteine group therein; c) contacting the sample with a second label adapted to bind to any reduced cysteine groups produced in step (b); and d) determining the ratio of the signal from the first label to the signal from the second label wherein the ratio indicates the redox status.
Claims42
A method for determining the redox status of a protein sample, the method comprising the steps of:
- a) contacting the sample with a first label adapted to bind to at least one reduced cysteine group therein;
- b) contacting the sample with a reducing agent to reduce at least one oxidised cysteine group therein;
- c) contacting the sample with a second label adapted to bind to any reduced cysteine groups produced in step (b);
- and
- d) determining the ratio of the signal from the first label to the signal from the second label wherein the ratio indicates the redox status.
A method for determining the redox status of a plurality of proteins in a sample, the method comprising the steps of:
- a) contacting the sample with a first label adapted to bind to at least one reduced cysteine group therein;
- b) contacting the sample with a reducing agent to reduce at least one oxidised cysteine group therein;
- c) contacting the sample with a second label adapted to bind to any reduced cysteine groups produced in step (b);
- d) determining the ratio of the signal from the first label to the signal from the second label wherein the ratio indicates the redox status;
- and
- e) compiling the results from (d) into a profile containing the details of the redox status of proteins in the sample.
A method according to any one of the preceding claims further comprising the pre-treatment step of protecting reduced cysteine groups in the sample prior to labelling.
A database containing a plurality of redox values for proteins obtained using the method of the invention.
A method for determining whether a protein sample has been modified by a protein modifying agent, the method comprising the steps of:
- a) contacting the sample with a first label adapted to bind to at least one reduced cysteine group therein;
- b) contacting the sample with a reducing agent to reduce at least one oxidised cysteine group therein;
- c) contacting the sample with a second label adapted to bind to any reduced cysteine groups produced in step (b);
- and
- d) determining the ratio of the signal from the first label to the signal from the second label and using said ratio to determine if the protein sample has been modified.
A method for determining whether a protein sample has been modified, the method comprising the steps of:
- a) contacting the sample with a first label adapted to bind to at least one reduced cysteine group therein;
- b) contacting the sample with a reducing agent to reduce at least one oxidised cysteine group therein;
- c) contacting the sample with a second label adapted to bind to any reduced cysteine groups produced in step (b);
- and
- d) determining the ratio of the signal from the first label to the signal from the second label and comparing the ratio to a reference ratio to determine if the sample has been modified.
A kit for treating a protein sample comprising:
- a) a first label adapted to bind to at least one reduced cysteine group in the protein sample;
- b) a reducing agent capable of reducing at least one oxidised cysteine group in the sample;
- and
- c) a second label adapted to bind to any reduced cysteine groups produced after use of the reagent in part (b).
A method for assessing a ROS associated pathology or disease in a subject, the method comprising the steps of
- a) contacting a protein sample from the subject with a first label adapted to bind to at least one reduced cysteine group therein;
- b) contacting the sample with a reducing agent to reduce at least one oxidised cysteine group therein;
- c) contacting the sample with a second label adapted to bind to any reduced cysteine groups produced in step (b);
- and
- d) determining the ratio of the signal from the first label to the signal from the second label and using said ratio to determine if the sample has been modified.
A method for assessing the efficacy of a therapeutic intervention for a ROS associated pathology or disease in a subject, the method comprising the steps of:
- a) contacting a protein sample from the subject with a first label adapted to bind to at least one reduced cysteine group therein;
- b) contacting the sample with a reducing agent to reduce at least one oxidised cysteine group therein;
- c) contacting the sample with a second label adapted to bind to any reduced cysteine groups produced in step (b);
- and
- d) determining the ratio of the signal from the first label to the signal from the second label and comparing said ratio obtained in the absence of the intervention with the ratio obtained in the presence of the intervention.
Dependencies are inferred from the claim text.