Polynucleotide Barcodes for Multiplexed Proteomics
Provided herein are methods for enhanced specificity of multiplexed measurements. Methods provided herein include immunoassay reactions and/or measuring protein-protein interactions with direct sequencing readouts of DNA barcodes.
1 . A complex comprising:
a substrate;
a first protein-binding moiety attached to the substrate;
a substrate polynucleotide barcode attached to the substrate or a substrate polynucleotide barcode attached to the first protein-binding moiety;
a second protein-binding moiety comprising a label;
wherein
the first protein-binding moiety and the second protein-binding moiety are both bound to an analyte;
and
an oligonucleotide hybridized to the substrate polynucleotide barcode, wherein the oligonucleotide comprises a labeled nucleotide.
2 . The complex of claim 1 , wherein (i) the first protein-binding moiety is a first aptamer, antibody, antigen-binding antibody fragment, or receptor, and (ii) the second protein-binding moiety is a second aptamer, antibody, antigen-binding antibody fragment, or receptor.
3 .- 6 . (canceled)
7 . The complex of claim 1 , wherein the second protein-binding moiety and the first protein-binding moiety are non-covalently bound to an analyte, wherein the analyte is a protein, an antibody, an antigen, or a ligand.
8 . (canceled)
9 . The complex of claim 1 , wherein the substrate is a bead.
10 . The complex of claim 1 , wherein the substrate polynucleotide barcode is 3-15 nucleotides in length.
11 . (canceled)
12 . The complex of claim 1 , wherein the first protein-binding moiety and the second protein-binding moiety are crosslinked.
13 . The complex of claim 1 , wherein the oligonucleotide comprising a labeled nucleotide is formed by incorporating with a polymerase a labeled nucleotide into a 3′-end of said oligonucleotide hybridized to the polynucleotide barcode.
14 .- 21 . (canceled)
22 . A method of detecting an analyte in a sample, the method comprising:
(a) contacting a substrate with the sample comprising an analyte, wherein said substrate comprises a first protein-binding moiety attached to the substrate, and (i) a substrate polynucleotide barcode attached to the substrate or (ii) a substrate polynucleotide barcode attached to the first protein-binding moiety; and binding said analyte to the first protein-binding moiety;
(b) contacting said substrate with a second protein-binding-moiety comprising a label attached to the second protein-binding moiety, and binding said analyte to the second protein-binding moiety, thereby forming the complex of claim 1 ,
(c) detecting the label attached to the second protein-binding moiety;
(d) detecting the oligonucleotide comprising the labeled nucleotide, thereby detecting the analyte.
23 . The method of claim 22 , wherein step (a) and step (b) are performed sequentially.
24 . (canceled)
25 . (canceled)
26 . (canceled)
27 . (canceled)
28 . The method of claim 22 , further comprising diagnosing a condition of a subject based on detecting the analyte.
29 . (canceled)
30 . The method of claim 22 , wherein the label comprises a protein polynucleotide barcode, and the method further comprises sequencing said protein polynucleotide barcode.
31 .- 37 . (canceled)
38 . The method of claim 30 , wherein the substrate polynucleotide barcode, the protein polynucleotide barcode, or both the substrate polynucleotide barcode and the protein polynucleotide barcode are 3-15 nucleotides in length.
39 .- 49 . (canceled)
50 . The method of claim 22 , further comprising crosslinking the first protein-binding moiety with the second protein-binding moiety, wherein the crosslinking comprises forming covalent bonds between the first protein-binding moiety, the analyte, and the second protein-binding moiety.
51 . (canceled)
52 . (canceled)
53 . The complex of claim 1 , wherein the label comprises a protein polynucleotide barcode.
54 . The complex of claim 53 , further comprising a second oligonucleotide hybridized to the protein polynucleotide barcode, wherein the second oligonucleotide complementary strand comprises a labeled nucleotide.
55 . The complex of claim 1 , wherein the label comprises a luciferase or a peroxidase.
56 . The complex of claim 1 , wherein the first protein-binding moiety is an antibody and the second protein binding moiety is an antibody.
57 . The complex of claim 1 , wherein the first protein-binding moiety and the second protein-binding moiety are independently IgA, IgD, IgE, IgG, or IgM.
58 . The complex of claim 1 , wherein the analyte is a protein or protein fragment.
59 . The complex of claim 1 , wherein the analyte is a hormone, cytokine, or glycoprotein.