Peptide constructs and assay systems

• US 9,958,454 B2
• Filed: 10/31/2013
• Issued: 05/01/2018
• Est. Priority Date: 04/08/2011
• Status: Active Grant

• Current Assignee: Prognosys Biosciences Inc.
• Original Assignee: Prognosys Biosciences Inc.
• Inventors: Kozlov, Igor A., Chee, Mark S., Capek, Petr, Routenberg, David A.
• Primary Examiner(s): QIAN, CELINE X
• Application Number:
  US14/068,921
• Publication Number:
  US 20140051589A1
• Time in Patent Office: 1,643 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1062 mRNA-Display, e.g. polypeptide and encoding template are connected covalently
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12Q 2521/537 Protease
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins

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.

Claims

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.

2. The method of claim 1, wherein the enzyme is a kinase and wherein the peptide portion of the at least one peptide construct is phosphorylated by the kinase.

3. The method of claim 2, wherein the kinase comprises a plurality of kinases.

4. The method of claim 2, wherein the enriching step is carried out by capturing the peptide portion that is phosphorylated by the kinase.

5. The method of claim 4, wherein the capturing is carried out by using an immobilized capture agent for the phosphorylated peptide portion.

6. The method of claim 5, wherein the capture agent comprises a phospho-specific antibody.

7. The method of claim 2, further comprising a step of labeling the peptide portion that is phosphorylated by the kinase with an affinity tag, wherein the affinity tag is capable of being captured by an immobilized capture agent specific for the affinity tag.

8. The method of claim 7, wherein the labeling step is catalyzed by the kinase.

9. The method of claim 2, wherein each peptide construct of the set further comprises an affinity tag capable of being captured by a capture agent specific for the affinity tag.

10. The method of claim 2, wherein in step (b) a single peptide construct comprising a peptide portion that is phosphorylated by the kinase is separated from peptide constructs comprising peptide portions that are not phosphorylated by the kinase.

11. The method of claim 2, wherein in step (b) a plurality of peptide constructs comprising peptide portions that are phosphorylated by the kinase are separated from peptide constructs comprising peptide portions that are not phosphorylated by the kinase.

12. The method of claim 2, further comprising a step of analyzing all or a portion of the identifying nucleic acid portions of the peptide constructs separated in step (b) comprising peptide portions that are not phosphorylated by the kinase, thereby identifying the peptide portions that are not phosphorylated by the kinase.

13. The method of claim 2, wherein the sequencing of step (d) comprises performing digital sequencing, nucleic acid sequencing, or any combination thereof.

14. The method of claim 1, wherein the enzyme is a protease and wherein the peptide portion of the at least one peptide construct is cleaved by the protease.

15. A method of assaying kinase activity in a sample, comprising:

(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.

16. The method of claim 15, wherein the kinase comprises a plurality of kinases.

17. The method of claim 15, wherein each peptide construct of the set further comprises an affinity tag capable of being captured by a capture agent specific for the affinity tag.

18. The method of claim 15, further comprising a step of analyzing all or a portion of the identifying nucleic acid portions of the peptide constructs separated in step (b) comprising peptide portions that are not phosphorylated by the kinase, thereby identifying the peptide portions that are not phosphorylated by the kinase.

19. The method of claim 15, wherein the sequencing of step (d) comprises performing digital sequencing, nucleic acid sequencing, or any combination thereof.

20. The method of claim 15, wherein the peptide portion of at least one of the peptide constructs is not phosphorylated by the kinase and is used as a control.

21. The method of claim 15, wherein:

the kinase is a first kinase;

in step (a), the peptide portion of the at least one peptide construct is a substrate for the first kinase, and the set further comprises at least one peptide construct of which the peptide portion is phosphorylated by a second kinase;

step (b) further comprises separating the at least one peptide construct comprising the peptide portion that is phosphorylated by the second kinase from peptide constructs comprising peptide portions that are not phosphorylated by the second kinase;

step (c) further comprises enriching the at least one peptide construct comprising the peptide portion that is phosphorylated by the second kinase; and

step (d) further comprises 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 second kinase, thereby identifying the peptide portion that is phosphorylated by the second kinase, wherein phosphorylation of the identified peptide portion by the second kinase indicates activity of the second kinase in the sample.

22. The method claim 21, further comprising comparing the peptide portion that is phosphorylated by the first kinase in the sample and the peptide portion that is phosphorylated by the second kinase in the sample.

23. The method of claim 15, wherein:

the sample is a first sample;

step (a) further comprises contacting a second sample with the set of peptide constructs, wherein the peptide portion of at least one of the peptide constructs is phosphorylated by a kinase in the second sample;

step (b) further comprises separating the at least one peptide construct comprising the peptide portion that is phosphorylated by the kinase in the second sample from peptide constructs comprising peptide portions that are not phosphorylated by the kinase in the second sample;

step (c) further comprises enriching the at least one peptide construct comprising the peptide portion that is phosphorylated by the kinase in the second sample; and

step (d) further comprises 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 in the second sample, thereby identifying the peptide portion that is phosphorylated by the kinase in the second sample, wherein phosphorylation of the identified peptide portion by the kinase indicates activity of the kinase in the second sample.

24. The method claim 23, further comprising comparing the peptide portion that is phosphorylated by the kinase in the first sample and the peptide portion that is phosphorylated by the kinase in the second sample.