Methods for balancing encoding signals of analytes
AssigneeEncodia, Inc.
InventorsDevin SULLIVAN, Norihito MURANAKA, Mark S. CHEE
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Abstract
The present disclosure relates to methods for high throughput analysis of analytes such as polypeptides in a cyclic manner (e.g., using NGPA or NGPS described herein) that allow adjustment of the dynamic range and sensitivity of analyte detection, for instance, based on the abundances of different polypeptides present in a sample to be analyzed. The approaches proposed herein can be used to adjust the dynamic range of abundant analytes (e.g., polypeptides present in high concentrations) in biological samples, such as plasma samples, and to increase proteome coverage achieved by high throughput protein analysis methods, for instance, by improving the sensitivity of detecting less abundant analytes.
Claims29
A method for analyzing a plurality of different polypeptides immobilized on a support, the method comprising:
- (a) contacting the plurality of different polypeptides comprising a first polypeptide and a second polypeptide with a binder, wherein the first polypeptide is associated with a first recording tag and the second polypeptide is associated with a second recording tag, and wherein the binder comprises:
- (i) a binding moiety capable of binding to the first polypeptide;
- and (ii) a handle attached to the binding moiety and configured to bind to or react with the first recording tag;
- (b) allowing the handle to bind to or react with the first recording tag brought in proximity by binding of the binder to the first polypeptide, thereby modifying the first recording tag to generate a modified first recording tag associated with the first polypeptide;
- (c) optionally, fragmenting the plurality of different polypeptides immobilized on the support to generate fragments of different polypeptides immobilized on the support;
- (d) contacting the plurality of different polypeptides or the fragments of different polypeptides with a plurality of binding agents, wherein each binding agent comprises:
- (i) a binding moiety capable of binding to a portion or component of a polypeptide of the plurality of different polypeptides or the fragments thereof;
- and (ii) a coding tag that comprises identifying information regarding the binding agent;
- (e) allowing transfer of identifying information from coding tags of the plurality of binding agents to recording tags associated with the plurality of different polypeptides or the fragments of different polypeptides, thereby generating an extended second recording tag associated with the second polypeptide or fragment thereof upon binding of a binding agent to the second polypeptide or fragment thereof, wherein transfer of identifying information to the modified first recording tag associated with the first polypeptide or fragment thereof is suppressed or blocked;
- and
- (f) analyzing the extended second recording tag to obtain identifying information regarding the binding agent that binds to the second polypeptide or fragment thereof, thereby obtaining information about the second polypeptide or fragment thereof, wherein the analyzing comprises nucleic acid sequencing.
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A method for analyzing molecules of a polypeptide immobilized on a support, the method comprising:
- (a) contacting the molecules of the polypeptide with a first binding agent and a second binding agent,
- wherein each molecule of the polypeptide is associated with a recording tag immobilized on a support,
- wherein the first binding agent comprises (i) a first binding moiety capable of binding to the polypeptide;
- and (ii) a first coding tag attached to the first binding moiety and comprising identifying information regarding the first binding agent, and
- wherein the second binding agent comprises (i) a second binding moiety capable of binding to the polypeptide;
- and, optionally, (ii) a handle attached to the second binding moiety and configured to bind to or react with the recording tag;
- (b) allowing transfer of the identifying information regarding the first binding agent from the first coding tag to the recording tag by primer extension and/or ligation to generate an extended recording tag, and optionally, allowing the handle to bind to or react with the first recording tag to generate a modified first recording tag;
- (c) contacting the molecules of the polypeptide with a third binding agent comprising (i) a third binding moiety capable of binding to the polypeptide;
- and (ii) a third coding tag attached to the third binding moiety and comprising identifying information regarding the third binding agent;
- (d) allowing transfer of the identifying information regarding the third binding agent from the third coding tag to the extended recording tag by primer extension and/or ligation to generate a further extended recording tag, wherein transfer of the identifying information regarding the third binding agent from the third coding tag to the recording tag or to the modified first recording tag is suppressed or blocked;
- and
- (e) analyzing the further extended recording tag to obtain identifying information regarding the first binding agent and/or the third binding agent, thereby obtaining information about the polypeptide, wherein the analyzing comprises nucleic acid sequencing.
Dependencies are inferred from the claim text.