Peptide constructs and assay systems

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.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]The present application claims priority to U.S. Ser. No. 61 / 473,709, filed Apr. 8, 2011.NOTICE OF GOVERNMENT FUNDING[0002]This invention was made with the support of the Federal Government under Grants GM090392; GM085884; and HG004284. The Federal Government may have rights in this invention.FIELD OF THE INVENTION[0003]This invention relates to methods of producing such sets of peptide constructs and methods of using the peptide constructs sets in assay systems and other analyses.BACKGROUND OF THE INVENTION[0004]In the following discussion certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.[0005]Rapid advances in DNA sequencing have creat...

AI Technical Summary

Benefits of technology

[0104]It is a distinct advantage of the invention that the individual peptides present in the peptide constructs of the invention can be detected through detection of the identifying nucleic acid, e.g., by sequencing the identifying nucleic acid (typically cDNA) associated with the peptide of interest in any particular peptide construct. The ability to identify the peptides in the peptide constructs of the invention by sequencing the identifying nucleic acids allows for very high throughput screening of the peptides using the cost effective mechanisms of sequencing, and is far more sensitive and scalable than direct peptide detection.

[0105]In one exemplary assay, the peptide constructs of the invention are used to test for protease activity. Generally, the peptide constructs used for determination of protease activity comprise identifying nucleic acids (cDNA or RNA molecules) attached to the C-terminus of the peptides of interest, with an affinity group (for example, a peptide capture tag or a biotin residue) attached at the N-terminus. When the peptide constructs are treated with a protease, peptides that are substrates for the protease will be cleaved and will lose the affinity group at the N-terminus. Therefore, only uncleaved peptides are captured with a capture moiety capable of binding to the N-terminal affinity tag. Employing such an affinity tag and capture moiety allows the peptide constructs having cleaved peptides to be separated from those with non-cleaved peptides. The identifying nucleic acids attached to the cleaved peptides are isolated and sequenced using highly-parallel, next-generation DNA sequencing. Alternatively, the nucleic acids with uncleaved peptides could be isolated and sequenced, though in some assays this would require many more constructs to be sequenced so in these cases it would be less preferred.

[0106]One embodiment of an exemplary protease assay is illustrated in FIG. 5. In this assay system, a pool of peptide constructs is synthesized, for example, by methods as shown in FIGS. 3 and / or 4. Each peptide construct 501 comprises a cDNA identifier 511 linked to a peptide comprising a test peptide 505 (the variable portion of the translated peptide), and a peptide sequence 503 that is a substrate for TEV protease, (namely, ENLYFQCA (SEQ ID NO:7)). Upon TEV protease cleavage at step 502, peptide sequence 503 is cleaved leaving an N-terminal cysteine-alanine (CA) 507 on each peptide construct. The N-terminal cysteine residue 507 is then modified with an affinity tag to allow capture on a solid support. In one preferred embodiment this is achieved using native chemical ligation by molecules containing thioesters, such as biotin-PEG-thioesters 513 (described in, e.g., Tolbert and Wong, Angew. Chem. Int. Ed., 41(12):2171-73 (2002)). The biotinylated peptides can be captured at step 504 using streptavidin-coated magnetic beads 515. At step 506, the captured peptide constructs are treated with a solution containing a protease of interest. Peptide constructs having peptides that are suitable substrates for the protease are cleaved and released from the streptavidin-coated magnetic beads. Alternatively, the protease cleavage reaction can be performed in solution before capture by the beads, followed by capture of uncleaved peptide constructs on magnetic beads. In either case, the uncleaved peptide constructs are immobilized on the beads and cleaved peptide constructs are released, facilitating separation of the two populations.

[0107]Numerous binding pairs can be used to separate reacted (transformed or modified) peptide constructs from unreacted (untransformed or unmodified) peptide constructs in the assays of the present invention. These include but are not limited to, streptavidin and short streptavidin binding peptides such as StrepTag (Schmidt, et al., J. Mol. Bio., 255:753-66 (1996); Schmidt and Skerra, J. Chromatog. A., 676:337-345 (1994); Skerra and Schmidt, Meth. in Enz., 326:271-304 (2000)), StrepTag II (Schmidt and Skerra, Nat. Protoc., 2:1528-35 (2007); Voss and Skerra, Protein Eng., 10(8):975-82 (1997)), and HPQ motifs (Gissel et al., J. of Peptide Science 1(4):217-226 (1995); Helms et al., JBC, 282(13):9813-24 (2007)); oligo histidine peptide tags and His6 binding groups (Kneusel et al., Procedures for the Analysis and Purification of His-tagged Proteins, in Nucleic Acid Protocols Handbook, p. 921 (2000) (Humana Press); Smith et al., Gene, 67:31-40 (1988)); FLAG peptide tags and His6 or His5 peptide groups (see, e.g., Kozlov, Combinatorial Chem. And High Throughput Screening, 11:24-35 (2008)); biotin and streptavidin, biotin and avidin, antibody-antigen pairs, and the like. Additionally, selective covalent linkage of peptide constructs to solid supports is possible. For example, N-terminal cysteine residues of cleaved peptides can be covalently coupled to thioester modified solid surfaces.

[0108]Alternatively, a chemically-reactive species (e.g., an aldehyde tag), label or other binding agent may be added in the construction of the peptide constructs. For example, introduction of a sulfatase consensus sequence recognized by the formylglycine-generating enzyme results in site-specific introduction of aldehyde groups into the peptide constructs. This consensus sequence can be between 6-13 amino acids, and the smallest such “aldehyde tags” are no larger than a His6 tag. Enzymatic modification at a sulfatase motif by formylglycine generating enzyme (FGE) generates a formylglycine (fGly) residue, which allows site-specific attachment of a capture agent or other moiety of interest to the peptide by covalent capture on hydrazine- or oxime-labeled oligo templates. This modification is reversible, and thus the introduction of this tag into the peptide constructs allows aldehyde-tagged peptides to be reversibly modified with multiple epitopes. Examples of aldehyde tags for use in the present invention are described in, e.g., US2008 / 0187956; Dierks and Frese, Chem. BioChem., 10:425-427 (2009); Wu, et al., www.pnas.org_cgi_doi—10.1073_pnas.807820106; Rush and Bertozzi, J. Am. Chem. Soc., 9:130:37, (2008); Landgrebe et al., Gene, 316: 47-56 (2003); Carrico, Nat. Chem. Biology, 3:6 (2007), each of which is incorporated by reference in its entirety for teaching useful tags and their use in peptide modification. Additionally, N-terminal formyl-methionine that is generated during translation initiation on all peptides can be specifically cleaved from peptides to expose the N-terminal cysteine. Two enzymes are required to remove formyl-methionine: peptide deformylase and methionine aminopeptidase. The resulting N-terminal cysteine residue can be used for peptide modification with an affinity residue (e.g., a biotin residue) or for direct immobilization on solid surfaces.

[0109]Returning to FIG. 5, the identifying nucleic acids (in this example, cDNAs) are then used as templates for amplification at step 508, with sequencing adaptors added during amplification. The amplified identifying nucleic acids are then sequenced at step 510, preferably using a next-generation DNA sequencing instrument. The sequence information obtained from sequencing the identifying nucleic acids (a) identifies which peptides were cleaved by the protease, and (b) provides information regarding the relative abundance of the cleaved peptides.

Problems solved by technology

However, these methods provide a random sampling of all possible n-mers, and are therefore inefficient for generating compact collections of protein sequences that are enriched for sequences of high biological relevance, such as peptides representing the human proteome.

Highly specific and sensitive high-throughput methods for assaying proteins as a large collection are also lacking.

Protein microarrays are a useful tool for such high throughput analysis of proteins, but the availability of microarray technology for large scale proteomics studies is still very limited due to the difficulty and cost of protein production (see Henderson and Bradley, Curr. Opin. Biotechnol., 18(4):326-30 (2007), Epub 2007 Aug. 6; and Tapia, Methods Mol. Biol., 570:3-17 (2009)).

However, the cost of generating arrays with tens of thousands or more spotted peptides is very high.

This is a major impediment to the use of large arrays of peptides for most applications, and severely limits accessibility of large arrays to researchers.

Several methods enable direct chemical synthesis of peptides in microarray format, which reduces costs, but these methods still have the major drawback of variability in the quality of the synthesized peptides (Antohe and Cooley, Methods Mol. Biol., 381:299-312 (2007)).

Moreover, the direct fabrication process can be very slow and inefficient (Hilpert, et al., Nat. Protoc., 2:1333-49 (2007)).

These approaches require individually synthesized nucleic acid templates, however, and the cost of these templates is higher than the cost of individual peptides arrayed by traditional methods.

In addition, analysis of the peptides is limited to substrate-based systems.

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