Peptide constructs and assay systems
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.
1. A method for analyzing a sample, comprising the steps of:
a. contacting the sample with a set of peptide constructs each comprising a peptide portion and an identifying nucleic acid portion,
wherein the peptide portion is encoded by an oligonucleotide sequence in the identifying nucleic acid portion,
wherein the set of peptide constructs comprises at least 5,000 distinct peptide constructs, and
wherein oligonucleotide sequences encoding peptide portions of the set of peptide constructs are custom-designed so that at least 10% of the set of peptide constructs contain contiguous peptide sequences of at least 12 amino acids that have more than 80% amino acid identity to protein sequences encoded in a genome of one or more species of organism,
b. separating a peptide construct whose peptide portion is acted upon by an agent in the sample from a peptide construct whose peptide portion is not acted upon by the agent, and
c. analyzing the identifying nucleic acid portion of the peptide construct whose peptide portion is acted upon by the agent or the identifying nucleic acid portion of the peptide construct whose peptide portion is not acted upon by the agent, thereby identifying the peptide portion that is or is not acted upon by the agent in the sample.
2. The method of claim 1 , wherein the peptide portion that is acted upon by the agent is modified permanently by the agent.
3. The method of claim 1 , wherein the analyzing step is performed by digital sequencing.
4. The method of claim 1 , wherein the analyzing step is performed by nucleic acid sequencing.
5. The method of claim 1 , wherein the set of peptide constructs comprises at least 100,000 distinct peptide constructs.
6. The method of claim 1 , wherein the set of peptide constructs comprises at least 1,000,000 distinct peptide constructs.
7. The method of claim 1 , wherein the one or more species of organism is of a eukaryotic species.
8. The method of claim 1 , wherein the one or more species of organism is of a mammalian species.
9. The method of claim 1 , wherein the one or more species of organism is of a bacterial species.
10. The method of claim 1 , wherein the one or more species of organism is a human pathogen.
11. The method of claim 1 , wherein the peptide portions of the set of peptide constructs comprise one or more sets of partially overlapping peptide sequences.
12. The method of claim 1 , wherein the peptide portion is dissociated from a ribosome before being linked to the identifying nucleic acid portion.
13. The method of claim 1 , wherein at least 90% of the oligonucleotide sequences encoding peptide portions of the set of peptide constructs contain no more than one in-frame codon that is a stop codon.
14. The method of claim 1 , further comprising first producing the oligonucleotide sequences encoding peptide portions of the set of peptide constructs by parallel synthesis.
15. The method of claim 1 , wherein the set of peptide constructs comprises at least 10,000 distinct peptide constructs.
16. The method of claim 1 , wherein the set of peptide constructs comprises at least 25,000 distinct peptide constructs.
17. The method of claim 1 , wherein the peptide portion that is acted upon by the agent is modified non-permanently by the agent.
18. The method of claim 1 , wherein the oligonucleotide sequences encoding peptide portions of the set of peptide constructs are custom-designed so that at least 10% of the set of peptide constructs contain contiguous peptide sequences of at least 12 amino acids that have more than 80% amino acid identity to protein sequences encoded in the genomes of up to 100 different species of organism.
19. The method of claim 1 , wherein the identifying nucleic acid portion comprises a promoter and a ribosomal binding site.
20. The method of claim 1 , wherein the identifying nucleic acid portion comprises a universal sequence or a sequence coding for a common peptide tag.
21. The method of claim 1 , wherein the analyzing step comprises sequencing the identifying nucleic acid portion of the peptide construct whose peptide portion is acted upon by the agent, thereby identifying the peptide portion that is acted upon by the agent in the sample.
22. The method of claim 1 , wherein the analyzing step comprises sequencing the identifying nucleic acid portion of the peptide construct whose peptide portion is not acted upon by the agent, thereby identifying the peptide portion that is not acted upon by the agent in the sample.
23. The method of claim 1 , wherein the set of peptide constructs is produced in a single reaction volume.
24. The method of claim 1 , wherein the set of peptide constructs is analyzed simultaneously in a single assay, and wherein the frequency of a peptide portion in the set of peptide constructs is obtained based on the frequency of the corresponding nucleic acid portion in a readout of step c.