Peptide constructs and assay systems
First Claim
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1. A method of identifying a substrate of an enzyme, comprising:
- (a) contacting a set of peptide constructs with an enzyme, wherein each peptide construct of the set comprises a peptide portion and an identifying nucleic acid portion that identifies the peptide portion, wherein the identifying nucleic acid portion of each peptide construct comprises a polynucleotide sequence or complement thereof encoding the peptide portion of the peptide construct, and wherein the peptide portion of at least one of the peptide constructs is acted upon by the enzyme;
(b) separating the at least one peptide construct comprising the peptide portion that is acted upon by the enzyme from peptide constructs comprising peptide portions that are not acted upon by the enzyme;
(c) enriching the at least one peptide construct comprising the peptide portion that is acted upon by the enzyme; and
(d) sequencing all or a portion of the identifying nucleic acid portion of the at least one peptide construct enriched in step (c) comprising the peptide portion that is acted upon by the enzyme, thereby identifying the peptide portion that is acted upon by the enzyme as a substrate for the enzyme.
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Abstract
The present invention provides methods for constructing peptide construct sets and methods of use of these peptide construct sets in assay systems for peptide analysis, and in particular for use in high throughput peptide analysis. The methods allow for analysis of large sets of peptide constructs in a cost-effective manner, employing molecular biological techniques that are both robust and easily parallelized. Thus, the methods allow for the construction of peptide construct sets encompassing, e.g., the human proteome.
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Citations
24 Claims
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1. A method of identifying a substrate of an enzyme, comprising:
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(a) contacting a set of peptide constructs with an enzyme, wherein each peptide construct of the set comprises a peptide portion and an identifying nucleic acid portion that identifies the peptide portion, wherein the identifying nucleic acid portion of each peptide construct comprises a polynucleotide sequence or complement thereof encoding the peptide portion of the peptide construct, and wherein the peptide portion of at least one of the peptide constructs is acted upon by the enzyme; (b) separating the at least one peptide construct comprising the peptide portion that is acted upon by the enzyme from peptide constructs comprising peptide portions that are not acted upon by the enzyme; (c) enriching the at least one peptide construct comprising the peptide portion that is acted upon by the enzyme; and (d) sequencing all or a portion of the identifying nucleic acid portion of the at least one peptide construct enriched in step (c) comprising the peptide portion that is acted upon by the enzyme, thereby identifying the peptide portion that is acted upon by the enzyme as a substrate for the enzyme. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A method of assaying kinase activity in a sample, comprising:
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(a) contacting a sample with a set of peptide constructs, wherein each peptide construct of the set comprises a peptide portion and an identifying nucleic acid portion that identifies the peptide portion, wherein the identifying nucleic acid portion of each peptide construct comprises a polynucleotide sequence or complement thereof encoding the peptide portion of the peptide construct, and wherein the peptide portion of at least one of the peptide constructs is phosphorylated by a kinase in the sample; (b) separating the at least one peptide construct comprising the peptide portion that is phosphorylated by the kinase from peptide constructs comprising peptide portions that are not phosphorylated by the kinase; (c) enriching the at least one peptide construct comprising the peptide portion that is phosphorylated by the kinase; and (d) sequencing all or a portion of the identifying nucleic acid portion of the peptide construct enriched in step (c) comprising the peptide portion that is phosphorylated by the kinase, thereby identifying the peptide portion that is phosphorylated by the kinase, wherein phosphorylation of the identified peptide portion by the kinase indicates activity of the kinase in the sample. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24)
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Specification