NEXT-GENERATION SEQUENCING FOR PROTEIN MEASUREMENT
Methods of detecting and quantifying target molecules, such as proteins, in a biological sample are provided. The disclosed methods include capturing target molecules with aptamers, replacing the aptamers with aptamer identification sequences, and then sequencing the aptamer identification sequences using next-generation sequencing techniques.
1 . A system for quantifying the abundances of target proteins in a biological sample, comprising:
a plurality of aptamers each configured to bind to a specific target protein upon exposure of a biological sample containing target proteins to the aptamers;
an aptamer-containing eluate containing the aptamers bound to the target proteins, wherein the aptamer-containing eluate is divided into a plurality of dilution groups each containing a particular concentration of aptamers based on the expected relative abundance of the aptamers in each dilution group;
a plurality of capture probes each configured to hybridize to a particular one of the aptamers, wherein each capture probe includes a DNA primer region and an aptamer ID sequence corresponding to the respective aptamer;
wherein the aptamer ID sequences are configured to be sequenced to determine the abundances of the target proteins in the biological sample; and
wherein the plurality of capture probes includes a plurality of distinct sets of capture probes, each set corresponding to the aptamers in one of the dilution groups.
2 . The system of claim 1 , wherein the plurality of dilution groups includes exactly two dilution groups.
3 . The system of claim 2 , wherein each of the two dilution groups is diluted by a ratio of 1:2, 1:4, or 1:16.
4 . The system of claim 1 , wherein the plurality of dilution groups includes exactly three dilution groups.
5 . The system of claim 4 , wherein each of the three dilution groups is diluted by a ratio of 1:2, 1:4, or 1:16.
6 . The system of claim 1 , wherein the plurality of dilution groups includes at least four dilution groups.
7 . The system of claim 6 , wherein each of the dilution groups is diluted by a ratio of 1:2, 1:4, or 1:16.
8 . A system for detecting target proteins in a biological sample, comprising:
a plurality of aptamers each configured to capture a specific protein;
a biological sample to which the aptamers may be exposed in order to capture one or more target proteins in the biological sample with corresponding aptamers;
an aptamer-containing eluate containing the aptamers bound to the target proteins, wherein the aptamer-containing eluate is divided into a plurality of dilution groups each containing a particular concentration of aptamers based on the expected relative abundance of the aptamers in each dilution group; and
a plurality of probes each including at least one DNA primer region and an aptamer ID sequence corresponding to one of the aptamers to which the probe is configured to be hybridized;
wherein the aptamer ID sequences are configured to be amplified and sequenced to identify the aptamer ID sequences, the aptamers that captured the target proteins, and the target proteins; and
wherein the plurality of probes includes a plurality of distinct sets of probes, each set corresponding to the aptamers contained in one of the dilution groups.
9 . The system of claim 8 , wherein the plurality of dilution groups includes exactly two dilution groups.
10 . The system of claim 9 , wherein each of the two dilution groups is diluted by a ratio of 1:2, 1:4, or 1:16.
11 . The system of claim 8 , wherein the plurality of dilution groups includes exactly three dilution groups.
12 . The system of claim 11 , wherein each of the three dilution groups is diluted by a ratio of 1:2, 1:4, or 1:16.
13 . The system of claim 8 , wherein the plurality of dilution groups includes at least four dilution groups.
14 . The system of claim 13 , wherein each of the dilution groups is diluted by a ratio of 1:2, 1:4, or 1:16.
15 . A system for quantifying the abundances of target proteins in a biological sample, comprising:
a plurality of aptamer-containing aliquots, each aliquot containing aptamers previously bound to a target protein after exposure of the aptamers to a plurality of target proteins in a biological sample, wherein the aliquots each contain a particular concentration of aptamers based on the expected relative abundance of the aptamers in each aliquot; and
a plurality of probes each including at least one DNA primer region and an aptamer ID sequence corresponding to one of the aptamers to which the probe is configured to be hybridized, wherein the aptamer ID sequences are configured to be amplified and sequenced to identify the aptamer ID sequences, the aptamers previously bound to the target proteins, and the target proteins; and
wherein the plurality of probes includes a plurality of distinct sets of probes, each set corresponding to the aptamers contained in one of the aliquots.
16 . The system of claim 15 , wherein the plurality of aliquots includes exactly two aliquots, and wherein each of the two aliquots is diluted by a ratio of 1:2, 1:4, or 1:16.
17 . The system of claim 15 , wherein the plurality of aliquots includes exactly three aliquots, and wherein each of the three aliquots is diluted by a ratio of 1:2, 1:4, or 1:16.
18 . The system of claim 15 , wherein the plurality of aliquots includes at least four aliquots, and wherein each of the aliquots is diluted by a ratio of 1:2, 1:4, or 1:16.
19 . The system of claim 15 , wherein at least one of the aliquots is diluted, and at least one of the aliquots is not diluted.
20 . The system of claim 19 , wherein each of the diluted aliquots is diluted by a ratio of 1:2, 1:4, or 1:16.