Compositions and methods for analyzing soluble proteins

Abstract

Provided herein is a method for analyzing post-translationally modified proteins. The method may include pre-enriching proteins using lectins and / or multiplexed detection of multiple proteins and / or multiple post-translational modifications. Also provided herein is a method for determining the likelihood that a subject has a disease or condition, such as cancer. The post-translationally modified proteins are derived from tumor cells. In some embodiments, the proteins to be assayed are derived from a subject who has or is suspected of having a disease or disorder, such as cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 376,047, filed September 16, 2022, which is incorporated by reference herein in its entirety for all purposes.

[0002] FIELD OF THE INVENTION The present disclosure provides compositions and methods related to analyzing post-translationally modified proteins in a sample. In some embodiments, the post-translationally modified proteins are derived from tumor cells. In some embodiments, the proteins to be assayed are derived from a subject who has or is suspected of having a disease or disorder, such as cancer. [Background technology]

[0003] Introduction and Abstract Protein post-translational modifications (PTMs) can affect protein activity and provide important information about abnormal conditions and pathologies. For example, cancer cells may express abnormally glycosylated proteins or abnormal levels of glycosylated proteins. However, it has been difficult to develop assays for rapidly and inexpensively measuring multiple post-translational modifications (e.g., multiple types of modifications and / or modifications of multiple different proteins) in a protein-specific manner. Summary of the Invention [Means for solving the problem]

[0004] The methods herein provide a means for quantifying and identifying levels of multiple PTMs to meet this need, to provide other benefits, or at least to provide the public with useful choices. Accordingly, the following illustrative examples are provided.

[0005] Embodiment 1 is as follows: 1. A method for analyzing post-translationally modified proteins in a sample, comprising: a) Pre-enrichment of post-translationally modified proteins, i) contacting the sample or sub-sample with a first lectin that specifically binds to a first saccharide present in a post-translational modification (PTM) on one or more target proteins in the sample, thereby generating a first complex comprising the first lectin and the target protein; and ii) separating the first complex from other components of the sample or sub-sample, thereby obtaining a first pre-enriched sub-sample. Including, pre-enriched and; b) determining the presence or level of at least one post-translationally modified target protein, i) contacting the first pre-enriched sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, wherein each of the first and second binding molecules comprises a label; and ii) detecting the labels of the first and second binding molecules. and A method comprising:

[0006] Embodiment 1.1 is as follows: 1. A method for analyzing post-translationally modified proteins in a sample, comprising: a) Pre-enrichment of post-translationally modified proteins, i) contacting the sample or sub-sample with a first binding unit that specifically binds to a first saccharide present in a post-translational modification (PTM) on one or more target proteins in the sample, thereby generating a first complex comprising the first binding unit and the target protein; and ii) separating the first complex from other components of the sample or sub-sample, thereby obtaining a first pre-enriched sub-sample. Including, pre-enriched and; b) determining the presence or level of at least one post-translationally modified target protein, i) contacting the first pre-enriched sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, wherein each of the first and second binding molecules comprises a label; and ii) detecting the labels of the first and second binding molecules. and A method comprising:

[0007] Embodiment 1.2 is as follows: The method of embodiment 1.1, wherein the binding unit can be an antibody, a lectin, or any molecule that specifically binds to a particular saccharide present in a post-translational modification (PTM) on one or more target proteins. In some embodiments, the antibody can be an anti-sialyl-Tn (STn) antibody.

[0008] Embodiment 1.3 is as follows: 1. A method for analyzing post-translationally modified proteins in a sample, comprising: a) Pre-enrichment of post-translationally modified proteins, i) contacting the sample or sub-sample with a plurality of lectins, the plurality of lectins including: (A) a first lectin that specifically binds to a first saccharide present in a post-translational modification (PTM) on one or more target proteins in the sample, thereby forming a first complex comprising the first lectin and the target proteins; and (B) a second lectin that specifically binds to a second saccharide present in the PTM on one or more target proteins, thereby forming a second complex comprising the second lectin and the target proteins; and ii) separating the first complex and the second complex from other components of the sample or sub-sample, thereby obtaining at least one pre-enriched sub-sample. Including, pre-enriched and; b) determining the presence or level of at least one post-translationally modified target protein, i) contacting at least one pre-enriched sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, wherein each of the first and second binding molecules comprises a label; and ii) detecting the labels of the first and second binding molecules. and A method comprising:

[0009] Embodiment 1.4 is as follows: The method of embodiment 1.3, comprising obtaining first and second pre-enriched sub-samples, wherein the second pre-enriched sub-sample comprises another component.

[0010] Embodiment 1.5 is the following: 1. A method for analyzing post-translationally modified proteins in a sample, comprising: a) Pre-enrichment of post-translationally modified proteins, i) contacting the sample or sub-sample with a first lectin that specifically binds to a first saccharide present in a post-translational modification (PTM) on one or more target proteins in the sample, thereby producing a first complex comprising the first lectin and the target protein, wherein the first lectin is in solution at the time of contacting; and ii) separating the first complex from other components of the sample or sub-sample, thereby obtaining a first pre-enriched sub-sample. Including, pre-enriched and; b) determining the presence or level of at least one post-translationally modified target protein, i) contacting the first pre-enriched sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, wherein each of the first and second binding molecules comprises a label; and ii) detecting the labels of the first and second binding molecules. and A method comprising:

[0011] Embodiment 1.6 is as follows: 1. A method for analyzing post-translationally modified proteins in a sample, comprising: a) Pre-enrichment of post-translationally modified proteins, i) contacting the sample or sub-sample with a first lectin that specifically binds to a first saccharide present in a post-translational modification (PTM) on one or more target proteins in the sample, thereby generating a first complex comprising the first lectin and the target protein; and ii) separating the first complex from other components of the sample or sub-sample, thereby obtaining a first pre-enriched sub-sample containing the first complex and a second sub-sample containing the other components. Including, pre-enriched and; b) determining the presence or level of at least one post-translationally modified target protein in at least a first pre-enriched sub-sample, and determining the presence or level of at least one target protein or post-translationally modified target protein in a second sub-sample, i) contacting a first pre-enriched sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, wherein each of the first and second binding molecules comprises a label; and contacting a second sub-sample with at least one of the first binding molecule and the second binding molecule; and ii) detecting the labels of the first and second binding molecules bound to the first target protein in the first pre-enriched sub-sample, and detecting at least one of the labels of the first and second binding molecules bound to the first target protein in the second sub-sample. and A method comprising:

[0012] Embodiment 1.7 is as follows: 1. A method for analyzing post-translationally modified proteins in a sample, comprising: a) Pre-enrichment of post-translationally modified proteins, i) contacting the sample or sub-sample with a first lectin that specifically binds to a first saccharide present in a post-translational modification (PTM) on one or more target proteins in the sample, thereby generating a first complex comprising the first lectin and the target protein; and ii) separating the first complex from other components of the sample or sub-sample, thereby obtaining a first pre-enriched sub-sample and a second sub-sample containing the other components. Including, pre-enriched and; b) determining the presence or level of at least one post-translationally modified target protein in at least a first pre-enriched sub-sample, and determining the presence or level of at least one target protein or post-translationally modified target protein in a second sub-sample, i) contacting a first pre-enriched sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, wherein the first and second binding molecules each comprise a label, and contacting a second sub-sample with at least a third binding molecule that binds to a third epitope of the target protein that is different from the first and second epitopes, wherein the third binding molecule comprises a label; and ii) detecting the labels of the first and second binding molecules bound to the first target protein in the first pre-enriched sub-sample, and detecting the label of the third binding molecule bound to the third epitope in the second sub-sample. and A method comprising:

[0013] Embodiment 1.8 is as follows: 8. The method of any one of embodiments 1.5-1.7, wherein pre-enriching comprises contacting the sample or one or more sub-samples thereof with a plurality of lectins, wherein the plurality of lectins comprises a first lectin and a second lectin that specifically binds to a second saccharide present on a PTM for one or more target proteins, wherein a second complex comprising the second lectin and the target protein is generated; and separating the first and second complexes from other components of the sample or one or more sub-samples thereof, thereby obtaining first and second pre-enriched sub-samples.

[0014] Embodiment 1.9 is as follows: 6. The method of any one of embodiments 1.3-1.5, wherein separating the first complex from other components of the sample or sub-sample thereof further comprises obtaining a second sub-sample comprising the other components.

[0015] Embodiment 1.10 is the following: step (b) determining the presence or level of at least one post-translationally modified target protein in at least a first pre-enriched sub-sample, and determining the presence or level of at least one target protein or post-translationally modified target protein in a second sub-sample; i) contacting at least one pre-enriched sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, wherein each of the first and second binding molecules comprises a label; and contacting a second sub-sample with at least one of the first binding molecule and the second binding molecule; and ii) detecting the labels of the first and second binding molecules bound to the first target protein in the first pre-enriched sub-sample, and detecting at least one of the labels of the first and second binding molecules bound to the first target protein in the second sub-sample. 4. The method of claim 1, further comprising the steps of:

[0016] Embodiment 1.11 is the following: step (b) determining the presence or level of at least one post-translationally modified target protein in at least a first pre-enriched sub-sample, and determining the presence or level of at least one target protein or post-translationally modified target protein in a second sub-sample; i) contacting at least one pre-enriched sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, wherein the first and second binding molecules each comprise a label, and contacting a second sub-sample with at least a third binding molecule that binds to a third epitope of the target protein that is different from the first and second epitopes, wherein the third binding molecule comprises a label; and ii) detecting the labels of the first and second binding molecules bound to the first target protein in the first pre-enriched sub-sample, and detecting the label of the third binding molecule bound to the third epitope in the second sub-sample. The method of embodiment 1.9, comprising the steps of:

[0017] Embodiment 1.12 is the following: The method of any one of embodiments 1.3-1.4 or 1.6-1.11, wherein the first lectin or lectins are in solution at the time of contacting.

[0018] Embodiment 1.13 is the following: 1. A method for analyzing post-translationally modified proteins in a sample, comprising: a) Pre-enrichment of post-translationally modified proteins, i) contacting the sample, or sub-sample thereof, with a plurality of lectins, the plurality of lectins including: (A) a first lectin that specifically binds to a first saccharide present in a post-translational modification (PTM) on one or more target proteins in the sample, thereby forming a first complex comprising the first lectin and the target proteins; and (B) a second lectin that specifically binds to a second saccharide present in the PTM on one or more target proteins, thereby forming a second complex comprising the second lectin and the target proteins, wherein the plurality of lectins are in solution upon contacting, and wherein at least the first and second lectins each include a label that comprises an oligonucleotide; and ii) separating the first complex and the second complex from other components of the sample or sub-sample, thereby obtaining at least one pre-enriched sub-sample. Including, pre-enriched and; b) determining the presence or level of at least one post-translationally modified target protein, i) contacting at least one pre-enriched sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, wherein the first and second binding molecules are antibodies, and each of the first and second binding molecules comprises a label; and ii) detecting the labels of the first and second binding molecules. and A method comprising:

[0019] Embodiment 1.14 is the following: The method of any one of embodiments 1.6 to 1.13, wherein the second sub-sample is a flow-through or a supernatant.

[0020] Embodiment 2 is as follows: 10. The method of any one of the preceding embodiments, wherein pre-enriching comprises contacting the sample or one or more sub-samples thereof with a plurality of lectins, wherein the plurality of lectins comprises a first lectin and a second lectin that specifically binds to a second saccharide present on a PTM for one or more target proteins, wherein a second complex comprising the second lectin and the target protein is generated; and separating the first and second complexes from other components of the sample or one or more sub-samples thereof, thereby obtaining first and second pre-enriched sub-samples.

[0021] Embodiment 3 is as follows: The method of any one of embodiments 1.3, 1.4, 1.8-1.4, or 2, wherein each of the lectins in the plurality of lectins specifically binds to a different saccharide.

[0022] Embodiment 4 is the following: 4. The method of any one of embodiments 1.4-3, wherein the pre-enrichment comprises parallel pre-enrichment comprising contacting a first sub-sample of the sample with a first lectin and contacting a second sub-sample of the sample with a second lectin.

[0023] Embodiment 5 is the following: 4. The method of any one of embodiments 1.4-3, wherein the pre-enrichment comprises sequential pre-enrichment comprising contacting the sample or sub-sample thereof with a first lectin, and separating the first complex from other components of the sample or sub-sample, thereby obtaining a first pre-enriched sub-sample and a first flow-through sub-sample comprising the other components of the sample or sub-sample, and contacting the first flow-through sub-sample with a second lectin, and separating the second complex from other components of the first flow-through sub-sample, thereby obtaining a second pre-enriched sub-sample.

[0024] Embodiment 6 is the following: 15. The method of any one of embodiments 1-1.14, wherein the pre-enrichment comprises simultaneously contacting the sample or sub-sample with multiple lectins, each lectin specifically binding to a different saccharide, wherein the multiple lectins comprise a first lectin and a second lectin that specifically binds to a second saccharide present on a PTM for one or more target proteins, wherein a second complex comprising the second lectin and the target protein is generated; and separating the first and second complexes from other components of the sample or one or more sub-samples, thereby obtaining at least a first pre-enriched sub-sample.

[0025] Embodiment 7 is the following: 10. The method of any one of the preceding embodiments, wherein the first epitope does not comprise a PTM or a portion of a PTM.

[0026] Embodiment 8 is the following: 7. The method of any one of embodiments 1 to 6, wherein the first epitope comprises a PTM or a portion of a PTM.

[0027] Embodiment 9 is the following: 10. The method of the immediately preceding embodiment, wherein the first epitope does not comprise the first saccharide or a portion of the first saccharide.

[0028] Embodiment 10 is the following: The method of any one of embodiments 1.13 to 6, wherein the first epitope does not comprise the first saccharide, a portion of the first saccharide, the second saccharide, or a portion of the second saccharide.

[0029] Embodiment 11 is the following: 11. The method of any one of embodiments 8-10, wherein the PTM of the first epitope, or portion thereof, comprises a saccharide, a phosphate moiety, a methyl moiety, an acetyl moiety, ubiquitin, a sumo moiety, a hydroxyl moiety, a lipid, or a nucleoside.

[0030] Embodiment 12 is the following: 10. The method of the immediately preceding embodiment, wherein the PTM of the first epitope, or portion thereof, comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide.

[0031] Embodiment 13 is the following: 10. The method of the immediately preceding embodiment, wherein the PTM, or portion thereof, of the first epitope comprises a monosaccharide, and optionally the monosaccharide is a GalNAc or Tn antigen.

[0032] Embodiment 14 is the following: 13. The method of embodiment 12, wherein the PTM of the portion of the first epitope comprises a tetrasaccharide, and optionally the tetrasaccharide is a sialyl Lewis saccharide.

[0033] Embodiment 15 is the following: 12. The method of embodiment 11, wherein the PTM of the first epitope, or portion thereof, comprises a methyl moiety.

[0034] Embodiment 16 is the following: 10. The method of the immediately preceding embodiment, wherein the first target protein is a histone.

[0035] Embodiment 17 is the following: 10. The method of any one of the preceding embodiments, wherein the second epitope does not comprise a PTM or a portion of a PTM.

[0036] Embodiment 18 is the following: 17. The method of any one of embodiments 1 to 16, wherein the second epitope comprises a PTM or a portion of a PTM.

[0037] Embodiment 19 is the following: The method of the immediately preceding embodiment, wherein the second epitope does not include the first saccharide or a portion of the first saccharide.

[0038] Embodiment 20 is the following: The method of any one of embodiments 1.14 to 16, wherein the second epitope does not comprise the first saccharide, a portion of the first saccharide, the second saccharide, or a portion of the second saccharide.

[0039] Embodiment 21 is the following: 21. The method of any one of embodiments 18-20, wherein the PTM or portion thereof of the second epitope comprises a saccharide, a phosphate moiety, a methyl moiety, an acetyl moiety, ubiquitin, a sumo moiety, a hydroxyl moiety, a lipid, or a nucleoside.

[0040] Embodiment 22 is the following: 10. The method of the immediately preceding embodiment, wherein the PTM, or portion thereof, of the second epitope comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide.

[0041] Embodiment 23 is the following: 10. The method of the immediately preceding embodiment, wherein the PTM, or portion thereof, of the second epitope comprises a monosaccharide, and optionally the monosaccharide is a GalNAc or Tn antigen.

[0042] Embodiment 24 is the following: 23. The method of embodiment 22, wherein the PTM, or portion thereof, of the second epitope comprises a tetrasaccharide, and optionally the tetrasaccharide is a sialyl Lewis saccharide.

[0043] Embodiment 25 is the following: 22. The method of embodiment 21, wherein the PTM of the second epitope, or portion thereof, comprises a methyl moiety.

[0044] Embodiment 26 is the following: 10. The method of the immediately preceding embodiment, wherein the first target protein is a histone.

[0045] Embodiment 27 is the following: 10. The method of any one of the preceding embodiments, wherein the plurality of binding molecules comprises a third binding molecule that specifically binds to a third epitope of the first target protein, the third binding molecule comprising a label, and wherein detecting comprises detecting the label of the third binding molecule.

[0046] Embodiment 28 is the following: 10. The method of claim 8, wherein the third epitope comprises a PTM or a portion of a PTM.

[0047] Embodiment 29 is the following: 29. The method of embodiment 27 or 28, wherein the third epitope does not include the first saccharide or a portion of the first saccharide.

[0048] Embodiment 30 is the following: 30. The method of any one of embodiments 27 to 29, wherein the third epitope does not comprise a saccharide that undergoes specific binding by the lectin used in the pre-enrichment step.

[0049] Embodiment 31 is the following: 31. The method of any one of embodiments 28-30, wherein the PTM or portion thereof of the third epitope comprises a saccharide, a phosphate moiety, a methyl moiety, an acetyl moiety, ubiquitin, a sumo moiety, a hydroxyl moiety, a lipid, or a nucleoside.

[0050] Embodiment 32 is the following: 10. The method of the immediately preceding embodiment, wherein the PTM, or portion thereof, of the third epitope comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide.

[0051] Embodiment 33 is the following: 10. The method of the immediately preceding embodiment, wherein the PTM or portion thereof of the third epitope comprises a monosaccharide, and optionally the monosaccharide is a GalNAc or Tn antigen.

[0052] Embodiment 34 is the following: 33. The method of embodiment 32, wherein the PTM, or portion thereof, of the third epitope comprises a tetrasaccharide, and optionally the tetrasaccharide is a sialyl Lewis saccharide.

[0053] Embodiment 35 is the following: 32. The method of embodiment 31, wherein the PTM of the third epitope, or portion thereof, comprises a methyl moiety.

[0054] Embodiment 36 is the following: 10. The method of the immediately preceding embodiment, wherein the third epitope is an epitope on a histone target protein.

[0055] Embodiment 37 is the following: 37. The method of any one of embodiments 27-36, wherein the plurality of binding molecules comprises a fourth binding molecule that specifically binds to a fourth epitope of the first target protein, the fourth binding molecule comprising a label, and wherein detecting comprises detecting the label of the fourth binding molecule.

[0056] Embodiment 38 is the following: 10. The method of claim 8, wherein the fourth epitope comprises a PTM or a portion of a PTM.

[0057] Embodiment 39 is the following: 39. The method of embodiment 37 or 38, wherein the fourth epitope does not include the first saccharide or a portion of the first saccharide.

[0058] Embodiment 40 is the following: 40. The method of any one of embodiments 37 to 39, wherein the fourth epitope does not comprise a saccharide that undergoes specific binding by the lectin used in the pre-enrichment step.

[0059] Embodiment 41 is the following: 41. The method of any one of embodiments 37-40, wherein the PTM of the fourth epitope, or portion thereof, comprises a saccharide, a phosphate moiety, a methyl moiety, an acetyl moiety, ubiquitin, a sumo moiety, a hydroxyl moiety, a lipid, or a nucleoside.

[0060] Embodiment 42 is the following: 10. The method of the immediately preceding embodiment, wherein the PTM, or portion thereof, of the fourth epitope comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide.

[0061] Embodiment 43 is the following: 10. The method of the immediately preceding embodiment, wherein the PTM or portion thereof of the fourth epitope comprises a monosaccharide, and optionally the monosaccharide is a GalNAc or Tn antigen.

[0062] Embodiment 44 is the following: 43. The method of embodiment 42, wherein the PTM, or portion thereof, of the fourth epitope comprises a tetrasaccharide, and optionally the tetrasaccharide is a sialyl Lewis saccharide.

[0063] Embodiment 45 is the following: 42. The method of embodiment 41, wherein the PTM of the fourth epitope, or portion thereof, comprises a methyl moiety.

[0064] Embodiment 46 is the following: 10. The method of the immediately preceding embodiment, wherein the fourth epitope is an epitope on a histone target protein.

[0065] Embodiment 47 is the following: 10. The method of any one of the preceding embodiments, wherein the plurality of binding molecules comprises a binding molecule comprising a label that specifically binds to an epitope of a second target protein that does not comprise a PTM or portion thereof, wherein the second target protein comprises a PTM, and wherein detecting comprises detecting the label of at least one binding molecule that specifically binds to the second target protein.

[0066] Embodiment 48 is the following: The method of the immediately preceding embodiment, wherein the second target protein comprises a PTM that is specifically bound by the first, second, third, or fourth binding molecule.

[0067] Embodiment 49 is the following: 49. The method of embodiment 47 or 48, wherein the plurality of binding molecules comprises at least one binding molecule that binds to an epitope of a second protein that comprises the PTM or a portion thereof.

[0068] Embodiment 50 is the following: 10. The method of any one of the preceding embodiments, comprising separating each lectin from each associated target protein of each of the complexes prior to contacting with the plurality of binding molecules.

[0069] Embodiment 51 is the following: 10. The method of any one of the preceding embodiments, wherein detection of the labels of the first and second binding molecules is used to quantify the first target protein in the sample or a sub-sample thereof.

[0070] Embodiment 52 is the following: A first sub-sample of the sample is contacted with a first lectin, and the method comprises: contacting an input sub-sample of the sample with a second plurality of binding molecules comprising the first binding molecule and a second binding molecule; and detecting the labels of the first and second binding molecules bound to the first target protein in the input subsample; 10. The method of any one of the preceding embodiments, further comprising:

[0071] Embodiment 53 is the following: The method of the immediately preceding embodiment, wherein detection of the labels of the first and second binding molecules bound to the first target protein in the input sub-sample is used to quantify the first target protein in the input sub-sample.

[0072] Embodiment 54 is the following: 54. The method of embodiment 52 or 53, wherein each of the plurality of target proteins is detected in the first sub-sample and in the input sub-sample using a plurality of labeled binding molecules specific for each of the plurality of target proteins.

[0073] Embodiment 55 is the following: 54. The method of embodiment 52 or 53, wherein each of the plurality of target proteins is quantified in the first sub-sample and in the input sub-sample using a plurality of labeled binding molecules specific for each of the plurality of target proteins.

[0074] Embodiment 56 is the following: 1. A method for analyzing post-translationally modified proteins in a sample, comprising: a) contacting the sample, or a sub-sample thereof, with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein, a second binding molecule that specifically binds to a second epitope of the first target protein, and a third binding molecule that specifically binds to a third epitope, wherein the third epitope is an epitope of the first target protein or an epitope of a second target protein, the first epitope comprises a PTM or a portion of a PTM that comprises a saccharide, each of the first, second, and third binding molecules comprises a label, and the first binding molecule comprises a lectin that specifically binds to the PTM; b) detecting the labels of the first, second, and third binding molecules; A method comprising:

[0075] Embodiment 56.1 is the following: 1. A method for analyzing post-translationally modified proteins in a sample, comprising: a) contacting the sample, or a sub-sample thereof, with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein, a second binding molecule that specifically binds to a second epitope of the first target protein, and a third binding molecule that specifically binds to a third epitope, wherein the third epitope is an epitope of the first target protein or an epitope of a second target protein, the first epitope comprises a PTM or a portion of a PTM that comprises a saccharide, each of the first, second, and third binding molecules comprises a label, and each of the first, second, and third binding molecules comprises a lectin that specifically binds to the PTM; b) detecting the labels of the first, second, and third binding molecules; A method comprising:

[0076] Embodiment 56.2 is the following: 1. A method for analyzing post-translationally modified proteins in a sample, comprising: a) contacting the sample, or sub-sample thereof, with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein, a second binding molecule that specifically binds to a second epitope of the first target protein, and a third binding molecule that specifically binds to a third epitope, wherein the third epitope is an epitope of the first target protein or an epitope of a second target protein, the first epitope comprises a PTM or a portion of a PTM that comprises a saccharide, each of the first, second, and third binding molecules comprises a label, the first binding molecule comprises a lectin that specifically binds to the PTM, and the first lectin is in solution at the time of contacting; b) detecting the labels of the first, second, and third binding molecules; A method comprising:

[0077] Embodiment 56.3 is the following: contacting the sample, or sub-sample thereof, with a plurality of binding molecules produces a first set of complexes comprising a first binding molecule and a first target protein, a second binding molecule and a first target protein, and a third binding molecule and either the first target protein or the second target protein; a) separating a first set of complexes from other components of the sample or sub-sample, thereby producing a first sub-sample comprising the first set of complexes and a second sub-sample comprising the other components; b) contacting the second sub-sample with one or more binding molecules, including a fourth binding molecule that comprises a label and that specifically binds to a fourth epitope; c) detecting the label of the fourth binding molecule bound to the fourth epitope in the second subsample; The method of embodiment 56.1 or 56.2, further comprising:

[0078] Embodiment 56.4 is the following: 1. A method for analyzing post-translationally modified proteins in a sample, comprising: a) contacting the sample, or sub-sample thereof, with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein, a second binding molecule that specifically binds to a second epitope of the first target protein, and a third binding molecule that specifically binds to a third epitope, wherein the third epitope is an epitope of the first target protein or an epitope of a second target protein, the first epitope comprises a PTM or a portion of a PTM that comprises a saccharide, each of the first, second, and third binding molecules comprises a label, and the first binding molecule comprises a lectin that specifically binds to the PTM, wherein contacting the sample, or sub-sample thereof, with the plurality of binding molecules produces a first set of complexes comprising the first binding molecule and the first target protein, the second binding molecule and the first target protein, and the third binding molecule and either the first target protein or the second target protein; a) separating a first set of complexes from other components of the sample or sub-sample, thereby creating a first sub-sample comprising the first set of complexes and a second sub-sample comprising the other components; b) contacting the second sub-sample with one or more binding molecules, the binding molecule comprising a label and a fourth binding molecule that specifically binds to a fourth epitope; c) detecting the labels of the first, second and third binding molecules in the first sub-sample and detecting the label of the fourth binding molecule in the second sub-sample; A method comprising:

[0079] Embodiment 56.5 is the following: The method of embodiment 56.3 or 56.4, wherein the fourth epitope is an epitope of the first target protein.

[0080] Embodiment 56.6 is the following: The method of embodiment 56.3 or 56.4, wherein the fourth epitope is an epitope of a second target protein.

[0081] Embodiment 56.7 is the following: The method of embodiment 56.3 or 56.4, wherein the fourth epitope is an epitope of a third target protein.

[0082] Embodiment 56.8 is the following: The method of any one of embodiments 56.3 to 56.7, wherein the second sub-sample is contacted with a plurality of binding molecules comprising a fourth binding molecule and a fifth binding molecule, and the fifth binding molecule specifically binds to a fifth epitope.

[0083] Embodiment 56.9 is the following: The method of embodiment 56.8, wherein the fifth epitope is an epitope of the first target protein.

[0084] Embodiment 56.10 is the following: The method of embodiment 56.8, wherein the fifth epitope is an epitope of a second target protein.

[0085] Embodiment 56.11 is the following: The method of embodiment 56.8, wherein the fifth epitope is an epitope of a third target protein.

[0086] Embodiment 56.12 is the following: The method of embodiment 56.8, wherein the fourth epitope is an epitope of a third target protein and the fifth epitope is an epitope of a fourth target protein.

[0087] Embodiment 57 is the following: The method of any one of embodiments 56 to 56.8, wherein the third epitope is an epitope of the first target protein and comprises a PTM or a portion of a PTM other than a saccharide or portion thereof that is specifically bound by the first epitope.

[0088] Embodiment 58 is the following: 58. The method of any one of embodiments 51-57, wherein the third epitope is an epitope of a second target protein, the plurality of binding molecules comprises a fourth binding molecule that specifically binds to a fourth epitope, the fourth epitope is an epitope of the second target protein, and the third epitope comprises a PTM or a portion of a PTM.

[0089] Embodiment 59 is the following: 59. The method of any one of embodiments 56-58, wherein the second epitope does not comprise a PTM.

[0090] Embodiment 60 is the following: 60. The method of embodiment 58 or 59, wherein the fourth epitope does not comprise a PTM.

[0091] Embodiment 61 is the following: 61. The method of any one of embodiments 56-60, wherein each PTM or portion thereof that is specifically bound by one of the plurality of binding molecules is independently selected from a saccharide, a phosphate moiety, a methyl moiety, an acetyl moiety, ubiquitin, a sumo moiety, a hydroxyl moiety, a lipid, or a nucleoside.

[0092] Embodiment 62 is the following: 10. The method of claim 8, wherein at least one PTM or portion thereof comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide.

[0093] Embodiment 63 is the following: The method of the immediately preceding embodiment, wherein at least one PTM or portion thereof comprises a monosaccharide, and optionally the monosaccharide is a GalNAc or Tn antigen.

[0094] Embodiment 64 is the following: 63. The method of embodiment 62, wherein at least one PTM, or portion thereof, comprises a tetrasaccharide, and optionally, the tetrasaccharide is a sialyl Lewis saccharide.

[0095] Embodiment 65 is the following: 62. The method of embodiment 61, wherein at least one PTM or portion thereof comprises a methyl moiety.

[0096] Embodiment 66 is the following: 10. The method of the immediately preceding embodiment, wherein at least one target protein is a histone.

[0097] Embodiment 67 is the following: 10. The method of any one of the preceding embodiments, wherein the lectin, or at least one of the lectins, specifically binds to a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide.

[0098] Embodiment 68 is the following: The method of the immediately preceding embodiment, wherein the lectin, or at least one of the lectins, specifically binds to a monosaccharide, and optionally the monosaccharide is a GalNAc or Tn antigen.

[0099] Embodiment 69 is the following: 68. The method of embodiment 67, wherein the lectin, or at least one of the lectins, specifically binds to a tetrasaccharide, and optionally the tetrasaccharide is a sialyl Lewis saccharide.

[0100] Embodiment 70 is the following: 10. The method of any one of the preceding embodiments, wherein at least one lectin and / or at least one binding molecule of the plurality of binding molecules is conjugated to a solid support.

[0101] Embodiment 71 is the following: The method of the immediately preceding embodiment, wherein the solid support comprises beads.

[0102] Embodiment 72 is the following: The method of the immediately preceding embodiment, wherein the solid support comprises magnetic beads.

[0103] Embodiment 73 is the following: 73. The method of any one of embodiments 70-72, wherein the first lectin is associated with a solid support.

[0104] Embodiment 74 is the following: 10. The method of any one of the preceding embodiments, wherein each label independently comprises a fluorophore, biotin, a peptide, or an oligonucleotide.

[0105] Embodiment 75 is the following: 10. The method of any one of the preceding embodiments, wherein each label comprises an oligonucleotide.

[0106] Embodiment 76 is the following: 10. The method of any one of the preceding embodiments, wherein detecting comprises a proximity ligation assay.

[0107] Embodiment 77 is the following: 76. The method of any one of embodiments 1-75, wherein detecting comprises a proximity extension assay.

[0108] Embodiment 78 is the following: 78. The method of any one of embodiments 75-77, wherein the label oligonucleotide of each binding molecule that specifically binds to an epitope of the first target protein each comprises a sequence that is complementary to the sequence of the label of at least one other binding molecule that specifically binds to an epitope of the first target protein.

[0109] Embodiment 79 is the following: 79. The method of any one of embodiments 75 to 78, wherein the labeled oligonucleotides of the first and second binding molecules comprise sequences that are complementary to each other.

[0110] Embodiment 80 is the following: 80. The method of any one of embodiments 75 to 79, wherein each labeled oligonucleotide comprises an adaptor.

[0111] Embodiment 81 is the following: 10. The method of claim 8, wherein each adapter comprises a barcode.

[0112] Embodiment 82 is the following: 82. The method of any one of embodiments 75-81, wherein detecting comprises amplifying labeled oligonucleotides that are hybridized to each other.

[0113] Embodiment 82.1 is the following: 83. The method of embodiment 82, wherein amplifying comprises quantitative amplification, optionally wherein the quantitative amplification comprises qPCR.

[0114] Embodiment 83 is the following: The method of embodiment 82 or 82.1, wherein detecting comprises sequencing the amplified oligonucleotides.

[0115] Embodiment 83.1 is the following: 84. The method of any one of embodiments 77 to 83, comprising a further amplification step after the proximity extension assay step, in which a barcode is attached to the oligonucleotide label, the barcode corresponding to the type of PTM pre-enriched during the pre-enrichment step.

[0116] Embodiment 83.2 is the following: The method of embodiment 83.1, wherein the barcode is a lectin type-specific barcode.

[0117] Embodiment 84 is the following: 76. The method of any one of embodiments 1-75, wherein the detection comprises an immunoassay.

[0118] Embodiment 85 is the following: The method of the immediately preceding embodiment, wherein the immunoassay is an enzyme-linked immunosorbent assay, a sandwich assay, an electrochemiluminescence assay, or a multiplex immunoassay.

[0119] Embodiment 86 is the following: 86. The method of embodiment 84 or 85, wherein detecting comprises flow cytometry analysis of the target protein.

[0120] Embodiment 87 is the following: 10. The method of any one of the preceding embodiments, comprising determining a level for one or more of the target proteins or for one or more PTM-containing versions of the target proteins based on the detection.

[0121] Embodiment 88 is the following: 10. The method of any one of the preceding embodiments, wherein the sample is obtained from a subject.

[0122] Embodiment 89 is the following: The method of the immediately preceding embodiment, wherein the sample is a blood sample.

[0123] Embodiment 90 is the following: The method of the immediately preceding embodiment, wherein the blood sample is a whole blood sample.

[0124] Embodiment 91 is the following: 90. The method of embodiment 89, wherein the blood sample is a plasma sample.

[0125] Embodiment 92 is the following: 90. The method of embodiment 89, wherein the blood sample is a plasma pellet sample or a buffy coat sample.

[0126] Embodiment 93 is the following: 56. The method of any one of embodiments 1-55, wherein at least one binding molecule of the plurality of binding molecules comprises a protein.

[0127] Embodiment 94 is the following: 10. The method of the immediately preceding embodiment, wherein at least one binding molecule of the plurality of binding molecules comprises a lectin other than any of the lectins used in the pre-enrichment step.

[0128] Embodiment 95 is the following: 10. The method of any one of the preceding embodiments, wherein at least one binding molecule in the plurality of binding molecules comprises a protein other than a lectin.

[0129] Embodiment 96 is the following: 10. The method of the immediately preceding embodiment, wherein at least one binding molecule of the plurality of binding molecules comprises VIM-1, or a methylcytosine-binding domain of VIM-1.

[0130] Embodiment 97 is the following: 10. The method of any one of the preceding embodiments, wherein at least one binding molecule of the plurality of binding molecules comprises an antibody.

[0131] Embodiment 98 is the following: 10. The method of any one of the preceding embodiments, wherein at least one binding molecule of the plurality of binding molecules comprises an aptamer.

[0132] Embodiment 99 is the following: 99. The method of any one of embodiments 1-98, wherein each of the plurality of binding molecules comprises a protein.

[0133] Embodiment 100 is as follows: 10. The method of claim 8, wherein each of the plurality of binding molecules comprises an antibody.

[0134] Embodiment 101 is as follows: 10. The method of any one of the preceding embodiments, wherein at least one target protein is a protein associated with a disease, or two or more of the plurality of target proteins are molecules associated with a disease, or each of the plurality of target proteins is a protein associated with a disease.

[0135] Embodiment 102 is the following: The method of the immediately preceding embodiment, wherein the disease is cancer.

[0136] Embodiment 103 is as follows: The method of the immediately preceding embodiment, wherein at least one target protein is upregulated in tumor cells relative to healthy cells of the same tissue type.

[0137] Embodiment 104 is the following: The method of any one of embodiments 101-103, wherein at least one, two or more, or each, of the target proteins is selected from RB1, TP53, PTEN, NF1, BRCA1, CEACAM1, CEACAM5, CEACAM6, EGFR, ErbB2, ErbB3, ErbB4, β-catenin, PD-L1, CTLA4, NYESO1, mesothelin, CA15-3, CA19-9, CA-125, CA27-29, and CA-72-4.

[0138] Embodiment 105 is the following: The method of any one of embodiments 101-104, wherein at least one target protein, two or more target proteins, or each of the plurality of target proteins is a cell type marker.

[0139] Embodiment 106 is the following: The method of the immediately preceding embodiment, wherein the cell type marker is selected from a marker of an immune cell and a solid tissue cell.

[0140] Embodiment 107 is the following: The method of the immediately preceding embodiment, wherein the cell type marker is selected from colon, lung, breast, skin, prostate, stomach, pancreatic markers and liver cell type markers.

[0141] Embodiment 108 is the following: The method of any one of any one of the preceding embodiments, comprising analyzing DNA in a sub-sample of the sample or in a second sample obtained from the same subject from which the first sample is obtained.

[0142] Embodiment 109 is the following: The method of the immediately preceding embodiment, wherein the sub-sample or second sample is a plasma or serum sample.

[0143] Embodiment 110 is as follows: The method of the immediately preceding embodiment, wherein the DNA is cfDNA. [Brief explanation of the drawings]

[0144] [Figure 1A-1] 1A-1B show an exemplary workflow of the methods disclosed herein for the analysis of PTMs. [Figure 1A-2] 1A-1B show an exemplary workflow of the methods disclosed herein for the analysis of PTMs. [Figure 1B-1] 1A-1B show an exemplary workflow of the methods disclosed herein for the analysis of PTMs. [Figure 1B-2] 1A-1B show an exemplary workflow of the methods disclosed herein for the analysis of PTMs.

[0145] [Figure 2] FIG. 2 is a schematic diagram of an example system suitable for use with some embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0146] Detailed Description of Certain Embodiments Reference will now be made in detail to certain specific embodiments of the invention. While the invention will be described in conjunction with such embodiments, it will be understood that it is not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the scope of the present invention as defined by the appended claims.

[0147] Before describing the present teachings in detail, it should be understood that the present disclosure is not limited to specific compositions or process steps, and therefore may vary.When used in this specification and the appended claims, it should be noted that the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.Thus, for example, reference to "a nucleic acid" includes plural nucleic acids, reference to "a cell" includes plural cells, etc.

[0148] Numerical ranges are inclusive of the numbers defining the range. It is understood that measured and measurable values ​​are approximate, taking into account significant digits and error associated with measurement. Also, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be limiting. It is to be understood that both the foregoing general and detailed descriptions are exemplary and explanatory only and are not restrictive of the present teachings.

[0149] Unless otherwise stated in the specification above, embodiments herein that recite "comprising" various components are also contemplated as "consisting of" or "consisting essentially of" the recited components, and embodiments herein that recite "consisting of" various components are also contemplated as "comprising" or "consisting essentially of" the recited components, and embodiments herein that recite "consisting essentially of" various components are also contemplated as "consisting of" or "comprising" the recited components (this interchangeability does not apply to the use of these terms in the claims).

[0150] The section headings used herein are for organizational purposes and should not be construed as limiting the disclosed subject matter in any way. In the event that any document or other material incorporated by reference contradicts any express content of this specification, including definitions, the present specification will control.

[0151] I. Definition "Post-translationally modified protein," as used herein, means a protein that has been covalently modified after translation by the attachment of an addendum (e.g., a phosphate, sugar, methyl, or acetyl moiety, etc.). Proteolysis and formation of disulfide bonds are not considered post-translational modifications.

[0152] "Lectin," as used herein, means a protein containing a non-immunoglobulin binding domain with specificity for carbohydrates (mono- or oligosaccharides).

[0153] "Epitope," as used herein, means that portion of a molecule or complex that is specifically bound by a binding molecule.

[0154] As used herein, "binding molecule" refers to a molecule that can specifically bind to an epitope. Binding molecules include, for example, nanobodies, aptamers, affimers, DARPins, lectins, and proteins, such as antibodies, that contain more than one polypeptide chain.

[0155] "Cell type marker," as used herein, means a molecule that is present in a higher proportion in one or more cell types than in other cell types present in the same sample, or than in any other cell type.

[0156] "Solid tissue cells," as used herein, refer to cells in or derived from solid tissue. Solid tissue cells exclude circulating cell types, e.g., cells normally found in blood or lymph. Examples of solid tissue cell types include, but are not limited to, colon, lung, breast, skin, prostate, stomach, pancreas, and liver cells.

[0157] "Cell-free DNA," "cfDNA molecule," or simply "cfDNA" refers to DNA molecules that naturally exist in a subject in an extracellular form (e.g., in blood, serum, plasma, or other bodily fluids, such as lymph, cerebrospinal fluid, urine, or sputum). cfDNA was previously present in the cell(s) of a large, complex biological organism, such as a mammal, but has been released from the cell(s) into the fluid found in the organism, and can be obtained from a sample of the fluid without the need for an in vitro cell lysis step. cfDNA molecules can exist as DNA fragments.

[0158] As used herein, a "blood sample" refers to a sample containing whole blood or components thereof (e.g., plasma, serum, buffy coat, plasma pellet).

[0159] As used herein, "partitioning" nucleic acids, such as DNA molecules, refers to separating, fractionating, or sorting a sample or population of nucleic acids into multiple subsamples or subpopulations of nucleic acids based on one or more modifications or characteristics that differ in proportion among the multiple subsamples or subpopulations. Partitioning can include physically dividing nucleic acid molecules based on the presence or absence of one or more methylated nucleic acid bases. A sample or population can be divided into one or more divided subsamples or subpopulations based on characteristics that indicate genetic or epigenetic changes or pathologies.

[0160] As used herein, the "originally isolated" form of a sample refers to the composition or chemical structure of the sample when it is isolated and before it is subjected to any procedure that alters the chemical structure of the isolated sample. Similarly, a feature "originally present" in a molecule refers to a feature that is present in the "original molecule" or in the molecule that "originally contains" the feature before the molecule is subjected to any procedure that alters the molecule's chemical structure.

[0161] As used herein, "base-pairing specificity" refers to the standard DNA base (A, C, G, or T) with which a given base most preferentially pairs. For example, unmodified cytosine and 5-methylcytosine have the same base-pairing specificity (i.e., specificity for G), whereas uracil and cytosine have different base-pairing specificities, with uracil having base-pairing specificity for A and cytosine having base-pairing specificity for G. Uracil's ability to form a wobble pair with G is not important, since uracil nevertheless most preferentially pairs with A among the four standard DNA bases.

[0162] As used herein, a "combination" containing multiple members refers to either a single composition containing the members or a set of compositions that are in close proximity, e.g., in separate containers or in compartments within a larger container such as a multi-well plate, test tube rack, refrigerator, freezer, incubator, water bath, ice bucket, machine, or other form of storage.

[0163] "Capturing" one or more target molecules, e.g., one or more proteins or nucleic acids or one or more molecules comprising at least one target region, refers to preferentially isolating or separating the one or more target molecules from non-target molecules.

[0164] As used herein, a "label" is a capture moiety, fluorophore, oligonucleotide or other moiety that facilitates detection, separation or isolation of what it is bound to.

[0165] As used herein, a "capture moiety" is a molecule that allows for affinity separation of a molecule linked to the capture moiety from a molecule lacking the capture moiety. Exemplary capture moieties include biotin, which allows for affinity separation by binding to streptavidin that is or can be linked to a solid phase, or an oligonucleotide, which allows for affinity separation by binding to a complementary oligonucleotide that is or can be linked to a solid phase.

[0166] As used herein, " tag " refers to a molecule or sequence that contains information that indicates the characteristics of the molecule with which the tag is associated. For example, a molecule can have a sample tag (distinguishing molecules in one sample from those in different samples), a molecular tag / molecular barcode / barcode (distinguishing different molecules from each other (both in unique tagging scenarios and non-unique tagging scenarios)), a distribution tag (distinguishing molecules in one distribution from molecules in a different distribution), or a purification tag.

[0167] As used herein, a "target protein" is a protein whose presence or absence is to be detected.

[0168] "Specifically binds," in the context of a binding molecule (e.g., a protein, primer, probe, or other oligonucleotide) and a target protein or sequence, means that, under appropriate binding conditions, the binding molecule binds to its target and forms a stable complex, while at the same time minimizing the formation of stable non-target complexes. For example, a primer or probe hybridizes to the target sequence or a copy thereof to a sufficiently greater extent than to non-target sequences, ultimately allowing capture or detection of the target sequence. Suitable binding conditions are well known in the art, can be predicted based on sequence composition, or can be determined by using routine testing methods (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) §§ 1.90-1.91, 7.37-7.57, 9.47-9.51, and 11.47-11.57, especially §§ 9.50-9.51, 11.12-11.13, 11.45-11.47, and 11.55-11.57, which are incorporated herein by reference).

[0169] "Substantially free" means free to a sufficient extent that the relevant property is not significantly affected by the presence of trace amounts of impurities.

[0170] "Immunoassay," as used herein, refers to an assay or method that involves contacting a molecule or sample with an antibody to test the function of one or more components of the sample or to detect, identify, and / or quantify the presence of a component. Examples of immunoassays include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs), sandwich assays, electrochemiluminescence (ECL) assays, and multiplex assays.

[0171] As used herein, the term "antibody" is used broadly to encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity.

[0172] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0173] A protein or nucleic acid is "tumor-produced" if it originates from a tumor cell. DNA originating from a tumor cell is "circulating tumor DNA" ("ctDNA"). Tumor cells are neoplastic cells that arise from a tumor, whether they remain within the tumor or detach from the tumor (e.g., in the case of metastatic cancer cells and circulating tumor cells).

[0174] "Target region," in the context of nucleic acids, refers to a genomic locus that is targeted for identification and / or capture, e.g., by using a probe (e.g., by sequence complementarity). A "target region set" or "set of target regions" refers to a plurality of genomic loci that are targeted for identification and / or capture, e.g., by using a set of probes (e.g., by sequence complementarity).

[0175] "Sequence variable target region" refers to a target region that may exhibit sequence changes, such as nucleotide substitutions (i.e., single-nucleotide variations), insertions, deletions, or gene fusions or rearrangements, in neoplastic cells (e.g., tumor and cancer cells) compared to normal cells. A sequence variable target region set is a set of sequence variable target regions. In some embodiments, the sequence variable target region is a target region that may exhibit changes affecting 50 or fewer consecutive nucleotides, e.g., 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer nucleotides.

[0176] "Epigenetic target region" refers to a target region that can show sequence-independent differences in different cell or tissue types (e.g., different types of immune cells) or neoplastic cells (e.g., tumor cells and cancer cells) compared to normal cells; or in DNA from different cell types or from subjects with cancer, e.g., cfDNA, compared to DNA from healthy subjects, e.g., cfDNA, or in cfDNA originating from different cell or tissue types that do not normally contribute substantially to cfDNA (e.g., immune, lung, colon, etc.), compared to background cfDNA (e.g., cfDNA derived from hematopoietic cells). Examples of sequence-independent changes include, but are not limited to, changes in methylation (increase or decrease), changes in nucleosome distribution, changes in cfDNA fragmentation patterns, changes in CCCTC-binding factor ("CTCF") binding, changes in transcription start site, and changes in regulatory protein binding regions. Therefore, epigenetic target region set includes, but is not limited to, hypermethylated variable target region set, hypomethylated variable target region set, and fragmented variable target region set, such as CTCF binding site and transcription start site.For this purpose, epigenetic target region set can also include the loci that are prone to neoplasia-related, tumor-related, or cancer-related local amplification and / or gene fusion, because, for example, the detection of copy number changes by sequencing or the detection of fusion sequences that map to more than one locus in a reference genome tends to be more similar to the detection of the exemplary epigenetic changes discussed above than the detection of nucleotide substitution, insertion, or deletion, in that the detection of local amplification and / or gene fusion does not depend on the accuracy of base calling at one or several individual positions, and therefore can be detected by relatively shallow sequencing depth.Epigenetic target region set is a set of epigenetic target regions.

[0177] The "capture yield" of a panel of probes for a given target set refers to the amount of nucleic acid corresponding to the target set that the panel of probes captures under typical conditions (e.g., the amount relative to another target set, or the absolute amount). Exemplary capture conditions are incubation of sample nucleic acid and probes in a small reaction volume (approximately 20 μL) containing a stringent hybridization buffer at 65°C for 10-18 hours. Capture yields may be expressed in absolute terms, or relative to multiple panels of probes. When capture yields for multiple sets of target regions are compared, the capture yield is normalized to the footprint size of the target region set (e.g., per kilobase). Thus, for example, if the footprint sizes of the first and second target regions are 50 kb and 500 kb, respectively (normalization factor 0.1), DNA corresponding to the first set of target regions will be captured with a higher yield than DNA corresponding to the second set of target regions when the mass per volume concentration of the captured DNA corresponding to the first set of target regions is greater than 0.1 times the mass per volume concentration of the captured DNA corresponding to the second set of target regions. As a further example, using the same footprint size, if the captured DNA corresponding to the first set of target regions has a mass per volume concentration that is 0.2 times the mass per volume concentration of the captured DNA corresponding to the second set of target regions, the DNA corresponding to the first set of target regions will be captured with a capture yield that is 2 times higher than the DNA corresponding to the second set of target regions.

[0178] The term "methylation" or "DNA methylation" refers to the addition of a methyl group to a nucleic acid base within a nucleic acid molecule. In some embodiments, methylation refers to the addition of a methyl group to a cytosine at a CpG site (cytosine-phosphate-guanine site (i.e., cytosine followed by guanine in the 5' to 3' direction of a nucleic acid sequence). In some embodiments, DNA ...CpG site)). In some embodiments, DNA methylation refers to the addition of a methyl group to a cytosine at a CpG site (cytosine-phosphate-guanine site (CpG site)). In some embodiments, DNA 6-methyladenine refers to the addition of a methyl group to adenine. In some embodiments, DNA methylation is 5-methylation (modification of the fifth carbon of the six-carbon ring of cytosine). In some embodiments, 5-methylation refers to the addition of a methyl group to the 5C position of cytosine, producing 5-methylcytosine (5mC). In some embodiments, methylation includes derivatives of 5mC. Derivatives of 5mC include, but are not limited to, 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC). In some embodiments, DNA methylation is 3C methylation (modification of the third carbon of the six-carbon ring of cytosine). In some embodiments, 3C methylation includes the addition of a methyl group to the 3C position of cytosine, producing 3-methylcytosine (3mC). Methylation can also occur at non-CpG sites; for example, methylation can occur at CpA, CpT, or CpC sites. DNA methylation can alter the activity of methylated DNA regions. For example, methylation of DNA within a promoter region can silence gene transcription. DNA methylation is crucial for normal development, and abnormal methylation can disrupt epigenetic regulation. Disruption of epigenetic regulation, for example, repression, can cause diseases such as cancer. Promoter methylation in DNA can indicate cancer.

[0179] The term "hypermethylated" refers to an increased level or degree of methylation of a nucleic acid molecule(s) within a population (e.g., a sample) of nucleic acid molecules relative to other nucleic acid molecules. In some embodiments, hypermethylated DNA can include DNA molecules containing at least one methylated residue, at least two methylated residues, at least three methylated residues, at least five methylated residues, or at least ten methylated residues.

[0180] The term "hypomethylated" refers to a reduced level or degree of methylation of a nucleic acid molecule(s) within a population (e.g., a sample) of nucleic acid molecules relative to other nucleic acid molecules. In some embodiments, hypomethylated DNA includes unmethylated DNA molecules. In some embodiments, hypomethylated DNA can include DNA molecules containing 0 methylated residues, up to 1 methylated residue, up to 2 methylated residues, up to 3 methylated residues, up to 4 methylated residues, or up to 5 methylated residues.

[0181] The term "agent that recognizes modified nucleobases in DNA," e.g., "agent that recognizes modified cytosine in DNA," refers to a molecule or reagent that binds to or detects one or more modified nucleobases in DNA, e.g., methylcytosine. A "modified nucleobase" is a nucleobase that contains a difference in chemical structure from an unmodified nucleobase. In the case of DNA, the unmodified nucleobase is adenine, cytosine, guanine, or thymine. In some embodiments, the modified nucleobase is a modified cytosine. In some embodiments, the modified nucleobase is a methylated nucleobase. In some embodiments, the modified cytosine is a methylcytosine, e.g., 5-methylcytosine. In such embodiments, the cytosine modification is methyl. Agents that recognize methylcytosine in DNA include, but are not limited to, "methyl-binding reagents," which, as used herein, refer to reagents that bind to methylcytosine. Methyl-binding reagents include, but are not limited to, methyl-binding domains (MBDs) and methyl-binding proteins (MBPs), and antibodies specific for methylcytosine. In some embodiments, such antibodies bind to 5-methylcytosine in DNA, which in some such embodiments may be single-stranded or double-stranded.

[0182] The terms "or a combination thereof" and "or a combination thereof," as used herein, refer to any and all permutations and combinations of the terms listed before the term combination. For example, "A, B, C, or a combination thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and, if order is important in the particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing this example, combinations containing repeats of one or more items or terms, e.g., BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are expressly included. Those skilled in the art will understand that, in general, there is no limit to the number of items or terms in any combination, unless otherwise clear from the context.

[0183] "Or" is used in its inclusive sense, ie, equivalent to "and / or" unless the context requires a different interpretation.

[0184] II. Exemplary Methods A. Overview; Target Protein; Lectin; Detection Steps In some embodiments, the methods described herein involve pre-enriching post-translationally modified proteins in a sample. Pre-enrichment is achieved by contacting the sample, or a sub-sample thereof, with a first lectin that specifically binds to a first saccharide present in a post-translational modification (PTM) on one or more target proteins in the sample, thereby generating a first complex comprising the first lectin and the target protein; and separating the first complex from other components of the sample, or sub-sample, thereby obtaining a first pre-enriched sub-sample. Multiple lectins can be used for such pre-enrichment, for example, simultaneously (whether performed in parallel on separate sub-samples or in a combined format on the same sample or sub-sample) or sequentially. See inset, parts a-c, of Figure 1A. In some embodiments, the plurality of lectins includes: (A) a first lectin that specifically binds to a first saccharide present in a post-translational modification on one or more target proteins in a sample, thereby generating a first complex comprising the first lectin and the target protein; and (B) a second lectin that specifically binds to a second saccharide present in a post-translational modification on one or more target proteins, thereby generating a second complex comprising the second lectin and the target protein. In some embodiments, the same target protein (e.g., the first target protein) comprises the first saccharide and the second saccharide. In other embodiments, different target proteins comprise the first saccharide and the second saccharide (e.g., the first target protein comprises the first saccharide and the second target protein comprises the second saccharide). The lower portion of Figure 1A illustrates a workflow in which two pre-enriched sub-samples are obtained in which different post-translationally modified proteins are enriched. The method may then include determining the presence or level of at least one of the post-translationally modified target proteins.This step can be achieved by contacting the first pre-enriched subsample with a plurality of binding molecules, including a first binding molecule that specifically binds to a first epitope of the first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, where each of the first and second binding molecules includes a label; and detecting the labels of the first and second binding molecules. Such detecting can be quantitative. In some embodiments, detecting includes determining the presence (or absence) or level of the labels of the first and second binding molecules.

[0185] Detection of the label can be performed in a manner that makes detection dependent on the proximity of the labels of the first and second binding molecules, such as a proximity ligation assay or a proximity extension assay. A workflow including a proximity extension assay is illustrated in FIG. 1A, in which, after extension, a further amplification is performed in which a barcode is added to the oligonucleotide label, the barcode corresponding to the type of pre-enriched PTM. In some embodiments, the barcode is a lectin-type-specific barcode, for example, as shown in FIG. 1A. The lectin-type-specific barcode can be used to identify a particular type of lectin, for example, a particular type of lectin used in a pre-enrichment step as disclosed herein (e.g., lectin X, Y, or Z, as illustrated in FIG. 1A). In such embodiments, each lectin used in the pre-enrichment step is specific for a particular PTM (e.g., each lectin specifically binds to a particular saccharide present on the PTM for one or more target proteins in the sample), and thus the lectin-type-specific barcode can be used to identify the PTM that has undergone binding by the lectin. These methods are beneficially applicable to higher order multiplexing because different labels can be used for different proteins and / or multiple lectins (or combinations of lectins and other binding molecules, e.g., for phosphate, methyl, acetyl, etc. PTMs) can be used to pre-enrich for multiple PTMs. Furthermore, the approach herein using enrichment followed by proximity assays (PLA or PEA) offers the potential for target multiplexing with high specificity using target-specific sandwich immunoassays. This is in contrast to PTM array approaches (direct immunoassays) that use a single binding molecule to detect a specific target protein.

[0186] In some embodiments, separating the first complex from other components of the sample or sub-sample thereof further comprises obtaining a second sub-sample that includes the other components. Such embodiments may further comprise determining the presence or level of at least one post-translationally modified target protein in at least a first pre-enriched sub-sample, and determining the presence or level of at least one target protein or post-translationally modified target protein in a second sub-sample, comprising: i) contacting at least one pre-enriched sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of the first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, wherein each of the first and second binding molecules comprises a label, and contacting the second sub-sample with at least one of the first binding molecule and the second binding molecule; and ii) detecting the label of the first and second binding molecules bound to the first target protein in the first pre-enriched sub-sample, and detecting at least one of the labels of the first and second binding molecules bound to the first target protein in the second sub-sample. In some embodiments, detecting comprises determining the presence (or absence) or level of at least one of the labels of the first and second binding molecules bound to the first target protein in the first sub-sample and / or the second sub-sample.

[0187] Some embodiments include determining the presence or level of at least one post-translationally modified target protein in at least a first pre-enriched sub-sample, and determining the presence or level of at least one target protein or post-translationally modified target protein in a second sub-sample, by: i) binding the at least one pre-enriched sub-sample to a plurality of binding molecules, the plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of the first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein; The method may further comprise the steps of: contacting the first and second binding molecules, each comprising a label, and contacting the second sub-sample with at least a third binding molecule that binds to a third epitope of the target protein different from the first and second epitopes, the third binding molecule comprising a label; and ii) detecting the labels of the first and second binding molecules bound to the first target protein in the first pre-enriched sub-sample, and detecting the label of the third binding molecule bound to the third epitope in the second sub-sample. In some embodiments, the detecting comprises determining the presence (or absence) or level of the first and second binding molecules bound to the first target protein in the first pre-enriched sub-sample, and determining the presence (or absence) or level of the third binding molecule bound to the third epitope in the second sub-sample.

[0188] In some embodiments of the disclosed methods, the first lectin or lectins are in solution at the time of contacting. A first lectin or lectins are considered to be in solution if they are not stably associated with a solid support, such as a bead, or the surface of a chip, well, array, or other solid object.

[0189] In some embodiments, the methods disclosed herein include pre-enrichment of post-translationally modified proteins comprising: i) contacting the sample, or a sub-sample thereof, with a first lectin that specifically binds to a first saccharide present in a post-translational modification (PTM) on one or more target proteins in the sample, thereby producing a first complex comprising the first lectin and the target protein; and ii) separating the first complex from other components of the sample, or sub-sample thereof, thereby obtaining the first pre-enriched sub-sample and a second sub-sample comprising the other components. Such embodiments further include determining the presence or level of at least one post-translationally modified target protein in at least a first pre-enriched sub-sample, and determining the presence or level of at least one target protein or post-translationally modified target protein in a second sub-sample, comprising: i) contacting the first pre-enriched sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of the first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, wherein each of the first and second binding molecules comprises a label, and contacting the second sub-sample with at least one of the first binding molecule and the second binding molecule; and ii) detecting the label of the first and second binding molecules bound to the first target protein in the first pre-enriched sub-sample, and detecting at least one of the labels of the first and second binding molecules bound to the first target protein in the second sub-sample.

[0190] In other embodiments, the methods disclosed herein include pre-enrichment of post-translationally modified proteins comprising: i) contacting the sample, or a sub-sample thereof, with a first lectin that specifically binds to a first saccharide present in a post-translational modification (PTM) on one or more target proteins in the sample, thereby producing a first complex comprising the first lectin and the target protein; and ii) separating the first complex from other components of the sample, or sub-sample, thereby obtaining a first pre-enriched sub-sample and a second sub-sample comprising the other components. Such embodiments include determining the presence or level of at least one post-translationally modified target protein in at least a first pre-enriched sub-sample, and determining the presence or level of at least one target protein or post-translationally modified target protein in a second sub-sample by: i) contacting the first pre-enriched sub-sample with a plurality of binding molecules, the plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of the first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein; and contacting the second sub-sample with at least a third binding molecule that binds to a third epitope of the target protein different from the first and second epitopes, the third binding molecule comprising a label; and ii) detecting the labels of the first and second binding molecules bound to the first target protein in the first pre-enriched sub-sample, and detecting the label of the third binding molecule bound to the third epitope in the second sub-sample.

[0191] In yet other embodiments, the methods disclosed herein include pre-enrichment of post-translationally modified proteins comprising: i) contacting a sample, or a sub-sample thereof, with a plurality of lectins, the plurality of lectins comprising: (A) a first lectin that specifically binds to a first saccharide present in a post-translational modification (PTM) on one or more target proteins in the sample, thereby generating a first complex comprising the first lectin and the target protein; and (B) a second lectin that specifically binds to a second saccharide present in the PTM on one or more target proteins, thereby generating a second complex comprising the second lectin and the target protein, wherein the plurality of lectins are in solution upon contacting, and at least the first and second lectins each comprise a label that comprises an oligonucleotide; and ii) separating the first complex and the second complex from other components of the sample, or sub-sample thereof, thereby obtaining at least one pre-enriched sub-sample. Such embodiments further include determining the presence or level of at least one post-translationally modified target protein, comprising: i) contacting at least one pre-enriched sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, wherein the first and second binding molecules are antibodies and each of the first and second binding molecules comprises a label; and ii) detecting the labels of the first and second binding molecules.

[0192] In some embodiments of the disclosed methods, the second sub-sample is the flow-through or the supernatant.

[0193] In some embodiments, the methods described herein include contacting a sample or subsample with a plurality of binding molecules, including a first binding molecule that specifically binds to a first epitope of a first target protein, a second binding molecule that specifically binds to a second epitope of the first target protein, and a third binding molecule that specifically binds to a third epitope of the first target protein. The third epitope can be an epitope of the first target protein or an epitope of the second target protein. The first epitope comprises a PTM or a portion of a PTM that includes a saccharide. Each of the first, second, and third binding molecules comprises a label, and the first binding molecule comprises a lectin that specifically binds to the PTM. The method further comprises detecting the labels of the first, second, and third binding molecules. This represents another approach for multiplexed detection of PTMs. The workflow of an exemplary embodiment of such a method is shown in Figure 1B. As exemplified therein, in some embodiments, multiple lectins (which may correspond to a first binding molecule, a third binding molecule, and / or one or more additional binding molecules) may be provided to bind different types of carbohydrate PTMs. In some embodiments, multiple binding molecules (e.g., antibodies) are provided to bind to a target protein (e.g., independently of PTMs). These may correspond to a second binding molecule, a third binding molecule, and / or one or more additional binding molecules. For clarity, the third binding molecule may, in different embodiments, bind to a PTM or to a target protein. The label of the binding molecule may comprise a nucleic acid that can be detected in a proximity-dependent manner, as discussed above. In this manner, the method can quantify the levels of multiple PTMs of one or more proteins. In embodiments in which two binding molecules are provided that bind to a given protein (independently of PTMs), the method can also quantify the protein independently of PTMs, and this can be multiplexed.

[0194] In some embodiments, a method described herein comprises: a) contacting a sample or a sub-sample thereof with a plurality of binding molecules, the plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein, a second binding molecule that specifically binds to a second epitope of the first target protein, and a third binding molecule that specifically binds to a third epitope, wherein the third epitope is an epitope of the first target protein or an epitope of a second target protein, the first epitope comprises a PTM or a portion of a PTM that comprises a saccharide, each of the first, second, and third binding molecules comprises a label, and each of the first, second, and third binding molecules comprises a lectin that specifically binds to the PTM; and b) detecting the labels of the first, second, and third binding molecules.

[0167] In other embodiments, a method described herein comprises the steps of: a) contacting a sample or sub-sample thereof with a plurality of binding molecules, comprising a first binding molecule that specifically binds to a first epitope of a first target protein, a second binding molecule that specifically binds to a second epitope of the first target protein, and a third binding molecule that specifically binds to a third epitope, wherein the third epitope is an epitope of the first target protein or an epitope of a second target protein, the first epitope comprises a PTM or a portion of a PTM that comprises a saccharide, each of the first, second, and third binding molecules comprises a label, the first binding molecule comprises a lectin that specifically binds to the PTM, and the first lectin is in solution at the time of contact; and b) detecting the labels of the first, second, and third binding molecules.

[0195] In yet other embodiments, the method comprises contacting the sample, or sub-sample thereof, with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein, a second binding molecule that specifically binds to a second epitope of the first target protein, and a third binding molecule that specifically binds to a third epitope, wherein the third epitope is an epitope of the first target protein or an epitope of a second target protein, the first epitope comprises a PTM or a portion of a PTM that comprises a saccharide, each of the first, second, and third binding molecules comprises a label, and the first binding molecule comprises a lectin that specifically binds to the PTM, wherein contacting the sample, or sub-sample thereof, with the plurality of binding molecules produces a first set of complexes comprising the first binding molecule and the first target protein, the second binding molecule and the first target protein, and the third binding molecule and either the first target protein or the second target protein. Such embodiments further comprise the steps of separating the first set of complexes from other components of the sample or sub-sample, thereby producing a first sub-sample comprising the first set of complexes and a second sub-sample comprising the other components; contacting the second sub-sample with one or more binding molecules comprising a label and a fourth binding molecule that specifically binds to a fourth epitope; and detecting the labels of the first, second and third binding molecules in the first sub-sample and detecting the label of the fourth binding molecule in the second sub-sample.

[0196] In some embodiments, the fourth epitope is an epitope of a first target protein. In other embodiments, the fourth epitope is an epitope of a second target protein. In other embodiments, the fourth epitope is an epitope of a third target protein.

[0197] In some embodiments, the second sub-sample is contacted with a plurality of binding molecules including a fourth binding molecule and a fifth binding molecule, wherein the fifth binding molecule specifically binds to a fifth epitope. In some embodiments, the fifth epitope is an epitope of a first target protein. In some embodiments, the fifth epitope is an epitope of a second target protein. In other embodiments, the fifth epitope is an epitope of a third target protein. In some embodiments, the fourth epitope is an epitope of a third target protein, and the fifth epitope is an epitope of a fourth target protein.

[0198] Unless the context clearly indicates otherwise, epitopes, e.g., a "first," "second," "third," "fourth," and / or "fifth" (or higher ordinal) epitope, are distinct from one another and may or may not be located on the same target protein. Similarly, unless the context clearly indicates otherwise, target proteins, e.g., but not limited to, a "first," "second," "third," "fourth," and / or "fifth" (or higher ordinal) target protein, are distinct from one another.

[0199] The contacting and detecting steps can be performed sequentially or simultaneously. In some embodiments, sequential methods include enriching, capturing, or isolating complexes comprising binding molecules (e.g., first and third, or second and third), and then detecting one or more target proteins. In some embodiments, the binding molecules (e.g., first, second, third, or additional binding molecules) are proteins that specifically bind to the PTM or target protein, such as antibodies, nanobodies, affimers, or DARpins. In some embodiments, the target molecule comprises a label, such as a capture moiety (e.g., biotin) or an oligonucleotide.

[0200] In some embodiments, the detecting step comprises contacting the sample with a binding molecule specific for a target protein suspected to be present in the sample. In some embodiments, the identity of one or more target proteins is known before commencing the method, and the target protein detection method is selected accordingly. In some embodiments, the detecting step comprises performing an immunoassay (e.g., an ELISA, a sandwich assay, an electrochemiluminescence (ECL) assay, or a multiplex immunoassay) in which one or more of the binding molecules is a lectin. In some embodiments, the detecting step comprises flow cytometric analysis of the sample.

[0201] In some embodiments, the one or more target proteins are derived from tumor cells, cells in another pathological condition, or cells that have been altered due to the presence of disease in the subject from which the cells are obtained. In some embodiments, the one or more target proteins are derived from a cell type that is not normally present in the type of body sample obtained from the subject.

[0202] In some embodiments, at least one target protein is a glycoprotein carbohydrate. In some embodiments, one or more target proteins are selected from RB1, TP53, PTEN, NF1, BRCA1, CEACAM1, CEACAM5, CEACAM6, EGFR, ErbB2, ErbB3, ErbB4, β-catenin, PD-L1, CTLA4, NYESO1, mesothelin, CA15-3, CA19-9, CA-125, CA27-29, and CA-72-4. In some embodiments, one or more target proteins include one or more target proteins known to exhibit altered post-translational modifications (e.g., altered glycosylation) in cancer. See, for example, Carman et al., AIMS Medical Science 3:386-416 (2016). In some embodiments, one or more target proteins are cell-type markers, such as immune cell-type markers or solid tissue cell-type markers. In some embodiments, the solid tissue cell type marker is a marker present in colon, lung, breast, skin, prostate, stomach, pancreatic or liver cells.

[0203] In some embodiments, one or more PTMs or portions thereof that are specifically bound by one of a plurality of binding molecules independently comprise a saccharide, a phosphate moiety, a methyl moiety, an acetyl moiety, a ubiquitin, a sumo moiety, a hydroxyl moiety, a lipid, or a nucleoside. In some embodiments, each PTM or portion thereof that is specifically bound by one of a plurality of binding molecules independently is selected from a saccharide, a phosphate moiety, a methyl moiety, an acetyl moiety, a ubiquitin, a sumo moiety, a hydroxyl moiety, a lipid, or a nucleoside. The saccharide can be a monosaccharide, disaccharide, trisaccharide, or tetrasaccharide, or a portion of a glycan, glycoprotein, or glycolipid. Exemplary saccharides include GalNAc, sialyl Lewis A, sialyl Lewis X, T antigen, and Tn antigen.

[0204] Lectins can be used in the methods disclosed herein. The selection of lectins will depend on the saccharide to be detected. Cancer cells may have altered N- and O-glycosylation processes. For example, abnormal O-glycans that may be expressed on the surface of cancer cells may exist as saccharide components of membrane-bound N-acetylgalactosamine (O-GalNAc) glycoproteins (T and Tn antigens) and glycolipids (Lewis a and Lewis x). Mucins, which are heavily O-GalNAc glycosylated proteins, are overexpressed by cancer cells and then secreted. See, for example, Poiroux et al., Int J Mol Sci. 2017 Jun; 18(6): 1232 (available at www.ncbi.nlm.nih.gov / pmc / articles / PMC5486055). Poiroux et al. and Ruiz-May et al., "N-Glycoprotein Enrichment by Lectin Affinity Chromatography" in Plant Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 1072, pages 633-643 (2013), each of which is incorporated herein by reference, discuss exemplary lectins useful for detecting various saccharides. In some embodiments, the lectin (e.g., the first lectin) in the methods described herein is a mannose-binding lectin, a fucose-binding lectin, a galactose- or N-acetylgalactosamine-binding lectin, or a sialic acid- or N-acetylglucosamine-binding lectin.In some embodiments, the lectin (e.g., the first lectin) in the methods described herein is concanavalin A (Con A), lentil lectin (LCH), snowdrop lectin (GNA), Ulex europaeus agglutinin (UEA), Aleuria aurantia lectin (AAL), Ricinus communis agglutinin (RCA), peanut agglutinin (PNA), jacalin (AIL), hairy vetch lectin (VVL), wheat germ agglutinin (WGA), elderberry lectin (SNA), or Maackia amurensis lectin (MAL).

[0205] In some embodiments, each label independently comprises a fluorophore, biotin, a peptide, or an oligonucleotide. In some embodiments, each label comprises an oligonucleotide. In some embodiments, at least the first lectin (and optionally the second lectin, third lectin, and / or fourth lectin) comprises a label comprising an oligonucleotide. In some embodiments, each of the plurality of lectins (e.g., the first, second, third, and / or fourth lectins) comprises a label comprising an oligonucleotide. In some embodiments, at least one binding molecule of the plurality of binding molecules comprises a lectin comprising a label comprising an oligonucleotide. In some embodiments, the first, second, and / or third binding molecule is a lectin comprising a label comprising an oligonucleotide. In embodiments, the oligonucleotide label of the first lectin can be the same as or different from the oligonucleotide label of the second, third, and / or fourth lectin. In some embodiments, each of the plurality of lectins (e.g., the first, second, third, and / or fourth lectins) comprises a label comprising an oligonucleotide, and each oligonucleotide label is the same. In other embodiments, each of the multiple lectins (e.g., the first, second, third and / or fourth lectins) comprises a label comprising an oligonucleotide, and the oligonucleotide label comprises 2, 3, 4, 5, 6, 7, 8, or more than 8 different oligonucleotides.

[0206] In some embodiments, each labeled oligonucleotide of each binding molecule that specifically binds to an epitope of a first target protein comprises a sequence complementary to the sequence of the labeled oligonucleotide of at least one other binding molecule that specifically binds to an epitope of the first target protein. In some embodiments, the labeled oligonucleotides of the first and second binding molecules comprise sequences complementary to each other. In some embodiments, each labeled oligonucleotide comprises an adapter (e.g., that can be used as a primer binding site for amplification and / or comprises a barcode). In some embodiments, the detecting step comprises amplifying the labeled oligonucleotides that hybridize to each other (e.g., as part of a proximity extension assay or a proximity ligation assay; in the latter case, the amplifying occurs after the ligating step). In some embodiments, the amplifying is quantitative, such as in qPCR. In some embodiments, the detecting step comprises sequencing the amplified oligonucleotides.

[0207] In some embodiments, one of the steps of detecting the label of the binding molecule comprises a proximity extension assay. In a proximity extension assay, first and second binding molecules targeting the same target protein or PTM and target protein are labeled with an oligonucleotide comprising a complementary hybridization sequence 3' to a tag (e.g., a molecular barcode that identifies the type of binding molecule with which the label is associated (e.g., a molecular barcode comprising a sequence unique to the type of binding molecule, e.g., a sequence unique to an antibody specific to a particular target protein), which may provide additional information, e.g., about the sample and / or pre-enriched fraction being analyzed; this may facilitate a subsequent pooling step). The tag may have any of the features described elsewhere herein for tags. When the oligonucleotides are in proximity (as occurs when the binding molecules are bound to the same target protein or target protein and its PTM), the hybridization sequences can hybridize to each other, thereby forming a substrate for extension by DNA polymerase. As a result, the extended product can be detected (e.g., by sequencing or qPCR, which may be performed after an amplification and / or library preparation step), thereby indicating the presence of the target protein or modified target protein in the sample or fraction.

[0208] In some embodiments, any of the steps of detecting the label of the binding molecule comprises a proximity ligation assay. In a proximity ligation assay, first and second binding molecules targeting the same target protein or PTM and target protein are labeled with oligonucleotides. A ligation template and a ligase are provided, so that ligation of the oligonucleotides occurs when they are in close proximity (as occurs when the binding molecules are bound to the same target protein or target protein and its PTM). The oligonucleotides may include tags or barcodes as discussed above and elsewhere herein. The tags may have any of the features described elsewhere herein for tags. The ligation product may be a substrate for amplification. The ligation product can be detected (e.g., by sequencing or qPCR, which may be performed after the amplification and / or library preparation step), thereby indicating the presence of the target protein or modified target protein in the sample or fraction.

[0209] In some embodiments, the detecting step comprises an immunoassay, such as an enzyme-linked immunosorbent assay, a sandwich assay, an electrochemiluminescence assay, or a multiplex immunoassay. In some embodiments, the detecting step comprises flow cytometry analysis of the target protein.

[0210] In some embodiments, the method includes determining a level for one or more of the target proteins or for one or more PTM-containing versions of the target proteins based on the detection.

[0211] In some embodiments, at least one binding molecule of the plurality of binding molecules comprises VIM-1 or a methylcytosine-binding domain of VIM-1. In some embodiments, at least one binding molecule of the plurality of binding molecules comprises an antibody. In some embodiments, at least one binding molecule of the plurality of binding molecules comprises an aptamer. In some embodiments, each of the plurality of binding molecules comprises a protein. In some embodiments, each of the plurality of binding molecules comprises an antibody.

[0212] In some embodiments, at least one target protein is a protein associated with a disease, or two or more of the multiple target proteins are molecules associated with a disease, or each of the multiple target proteins is a protein associated with a disease, such as cancer.In some embodiments, at least one target protein is post-translationally modified differentially in tumor cells compared with healthy cells of the same tissue type.In some embodiments, at least one target protein is up-regulated in tumor cells compared with healthy cells of the same tissue type.

[0213] In some embodiments, at least one target protein, two or more target proteins, or each of the plurality of target proteins is a cell type marker. In some embodiments, the cell type marker is selected from a marker for immune cells and solid tissue cells. In some embodiments, the cell type marker is selected from a marker for colon, lung, breast, skin, prostate, stomach, pancreas, and liver cell type marker.

[0214] In some embodiments, the method comprises analyzing the DNA in a sub-sample of the sample or in a second sample obtained from the same subject from which the first sample is obtained.The sub-sample or the second sample can be plasma or serum sample.The DNA can be cfDNA.

[0215] In some embodiments, the detecting step facilitates the diagnosis of disease or the identification of appropriate treatment. In some embodiments, the presence or altered levels of one or more target proteins indicates the presence of a disease or disorder in the subject, such as cancer, a precancerous condition, an infectious disease, transplant rejection, or other disorder that causes altered cell death. In some embodiments, detecting the label of the binding molecule in combination with cfDNA analysis of sequence alterations in sequence-variable target regions and / or sequence-independent alterations in epigenetic target regions, e.g., cfDNA analysis as described herein, indicates the presence of a disease or disorder in the subject, such as cancer, a precancerous condition, an infectious disease, transplant rejection, or other disorder that causes altered DNA alterations and altered relative amounts of PTMs of target proteins compared to healthy subjects.

[0216] Pre-enrichment may include contacting the sample, or one or more sub-samples thereof, with a plurality of lectins, wherein the plurality of lectins includes a first lectin and a second lectin that specifically binds to a second saccharide present on a PTM for one or more target proteins, such that a second complex comprising the second lectin and the target protein is generated; and separating the first and second complexes from other components of the sample, or one or more sub-samples thereof, thereby obtaining first and second pre-enriched sub-samples. In some embodiments, each lectin in the plurality of lectins specifically binds to a different saccharide. In some embodiments, pre-enrichment includes parallel pre-enrichment, comprising contacting a first sub-sample of the sample with the first lectin and contacting a second sub-sample of the sample with the second lectin. In some embodiments, pre-enrichment comprises sequential pre-enrichment comprising contacting the sample or sub-sample thereof with a first lectin, and separating the first complex from other components of the sample or sub-sample, thereby obtaining a first pre-enriched sub-sample and a first flow-through sub-sample comprising the other components of the sample or sub-sample, and contacting the first flow-through sub-sample with a second lectin, and separating the second complex from other components of the first flow-through sub-sample, thereby obtaining a second pre-enriched sub-sample.

[0217] In some embodiments, pre-enrichment comprises simultaneously contacting the sample or sub-sample thereof with multiple lectins, each lectin specifically binding to a different saccharide, wherein the multiple lectins comprise a first lectin and a second lectin that specifically binds to a second saccharide present on a PTM for one or more target proteins, such that a second complex comprising the second lectin and the target protein is generated; and separating the first and second complexes from other components of the sample or one or more sub-samples thereof, thereby obtaining at least a first pre-enriched sub-sample.

[0218] The first epitope may or may not include a PTM or a portion of a PTM. In some embodiments, the first epitope does not include the first saccharide or a portion of the first saccharide. In some embodiments, the first epitope does not include the first saccharide, a portion of the first saccharide, a second saccharide, or a portion of the second saccharide.

[0219] In some embodiments, the PTM of the first epitope, or a portion thereof, comprises a saccharide, a phosphate moiety, a methyl moiety, an acetyl moiety, a ubiquitin, a sumo moiety, a hydroxyl moiety, a lipid, or a nucleoside. For example, the PTM of the first epitope, or a portion thereof, can comprise a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide. In some embodiments, the PTM of the first epitope, or a portion thereof, comprises a monosaccharide, optionally the monosaccharide is a GalNAc or Tn antigen. In some embodiments, the PTM of the first epitope, or a portion thereof, comprises a tetrasaccharide, optionally the tetrasaccharide is a sialyl Lewis saccharide. In some embodiments, the PTM of the first epitope, or a portion thereof, comprises a methyl moiety. In some embodiments, the PTM of the first epitope, or a portion thereof, is that of a histone.

[0220] The second epitope may or may not include a PTM or a portion of a PTM. In some embodiments, the second epitope does not include the first saccharide or a portion of the first saccharide. In some embodiments, the second epitope does not include the first saccharide, a portion of the first saccharide, the second saccharide, or a portion of the second saccharide.

[0221] In some embodiments, the second epitope PTM or portion thereof comprises a saccharide, a phosphate moiety, a methyl moiety, an acetyl moiety, a ubiquitin, a sumo moiety, a hydroxyl moiety, a lipid, or a nucleoside. In some embodiments, the second epitope PTM or portion thereof comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide. In some embodiments, the second epitope PTM or portion thereof comprises a monosaccharide, optionally the monosaccharide is a GalNAc or Tn antigen. In some embodiments, the second epitope PTM or portion thereof comprises a tetrasaccharide, optionally the tetrasaccharide is a sialyl Lewis saccharide. In some embodiments, the second epitope PTM or portion thereof comprises a methyl moiety. In some embodiments, the second epitope PTM or portion thereof is that of a histone.

[0222] In some embodiments, the plurality of binding molecules includes a third binding molecule that specifically binds to a third epitope of the first target protein, the third binding molecule including a label, and the detecting step includes detecting the label of the third binding molecule. In some embodiments, the third epitope includes a PTM or a portion of a PTM. In some embodiments, the third epitope does not include the first saccharide or a portion of the first saccharide. In some embodiments, the third epitope does not include a saccharide that is specifically bound by the lectin used in the pre-enrichment step. In some embodiments, the PTM of the third epitope, or a portion thereof, includes a saccharide, a phosphate moiety, a methyl moiety, an acetyl moiety, a ubiquitin, a sumo moiety, a hydroxyl moiety, a lipid, or a nucleoside. In some embodiments, the PTM of the third epitope, or a portion thereof, includes a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide. In some embodiments, the third epitope PTM or portion thereof comprises a monosaccharide, and optionally the monosaccharide is a GalNAc or Tn antigen. In some embodiments, the third epitope PTM or portion thereof comprises a tetrasaccharide, and optionally the tetrasaccharide is a sialyl Lewis saccharide. In some embodiments, the third epitope PTM or portion thereof comprises a methyl moiety. In some embodiments, the third epitope is an epitope on a histone target protein.

[0223] In some embodiments, the plurality of binding molecules includes a fourth binding molecule that specifically binds to a fourth epitope of the first target protein, the fourth binding molecule including a label, and the detecting step includes detecting the label of the fourth binding molecule. In some embodiments, the fourth epitope includes a PTM or a portion of a PTM. In some embodiments, the fourth epitope does not include the first saccharide or a portion of the first saccharide. In some embodiments, the fourth epitope does not include a saccharide that is specifically bound by the lectin used in the pre-enrichment step. In some embodiments, the PTM of the fourth epitope, or a portion thereof, includes a saccharide, a phosphate moiety, a methyl moiety, an acetyl moiety, ubiquitin, a sumo moiety, a hydroxyl moiety, a lipid, or a nucleoside. In some embodiments, the PTM of the fourth epitope, or a portion thereof, includes a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide. In some embodiments, the fourth epitope PTM or portion thereof comprises a monosaccharide, and optionally the monosaccharide is a GalNAc or Tn antigen. In some embodiments, the fourth epitope PTM or portion thereof comprises a tetrasaccharide, and optionally the tetrasaccharide is a sialyl Lewis saccharide. In some embodiments, the fourth epitope PTM or portion thereof comprises a methyl moiety. In some embodiments, the fourth epitope is an epitope on a histone target protein.

[0224] In some embodiments, the plurality of binding molecules comprises binding molecules comprising a label that specifically binds to an epitope of a second target protein that does not comprise a PTM or a portion thereof, wherein the second target protein comprises a PTM, and the detecting step comprises detecting the label of at least one binding molecule that specifically binds to the second target protein. In some embodiments, the second target protein comprises a PTM that is specifically bound by the first, second, third, or fourth binding molecule. In some embodiments, the plurality of binding molecules comprises at least one binding molecule that binds to an epitope of the second protein that comprises a PTM or a portion thereof.

[0225] In some embodiments, the method comprises separating each lectin from each associated target protein of the complex prior to contacting with the plurality of binding molecules.

[0226] In some embodiments, the third epitope is an epitope of the first target protein and comprises a PTM or a portion of a PTM other than the saccharide or portion thereof that is specifically bound by the first epitope.

[0227] In some embodiments, the third epitope is an epitope of a second target protein, the plurality of binding molecules comprises a fourth binding molecule that specifically binds to a fourth epitope, the fourth epitope is an epitope of the second target protein, and the third epitope comprises a PTM or a portion of a PTM.

[0228] In some embodiments, the second epitope does not comprise a PTM. In some embodiments, the fourth epitope does not comprise a PTM.

[0229] In some embodiments, each PTM or portion thereof that is specifically bound by one of the plurality of binding molecules is independently selected from a saccharide, a phosphate moiety, a methyl moiety, an acetyl moiety, a ubiquitin, a sumo moiety, a hydroxyl moiety, a lipid, or a nucleoside. In some embodiments, at least one PTM or portion thereof comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide. In some embodiments, at least one PTM or portion thereof comprises a monosaccharide, optionally wherein the monosaccharide is a GalNAc or Tn antigen. In some embodiments, at least one PTM or portion thereof comprises a tetrasaccharide, optionally wherein the tetrasaccharide is a sialyl Lewis saccharide. In some embodiments, at least one PTM or portion thereof comprises a methyl moiety. In some embodiments, at least one target protein is a histone.

[0230] In some embodiments, the lectin, or at least one of the lectins, specifically binds to a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide. In some embodiments, the lectin, or at least one of the lectins, specifically binds to a monosaccharide, optionally the monosaccharide is a GalNAc or Tn antigen. In some embodiments, the lectin, or at least one of the lectins, specifically binds to a tetrasaccharide, optionally the tetrasaccharide is a sialyl Lewis saccharide.

[0231] Solution-based approaches (e.g., where the lectins disclosed herein are in solution, i.e., not bound to a solid support, at the time of contacting) may provide enhanced assay sensitivity (e.g., enhanced detection of rare modifications) compared to array-based approaches (e.g., where the lectins are bound to a solid support). Solution-based approaches may provide more opportunity (compared to array-based approaches) for the disclosed lectins to interact with (e.g., bind to) target molecules, since in such approaches the lectins are free to diffuse throughout the sample. Thus, in some embodiments, the first lectin (and optionally one or more additional lectins, e.g., a second lectin and / or a third lectin) is in solution when the sample or sub-sample thereof is contacted with the first (and optionally the second, third, etc.) lectin. In such embodiments, the lectin is not bound to a solid substrate, e.g., a bead or array surface, e.g., when the sample or sub-sample thereof is contacted. Additionally, the use of oligonucleotide labels may further enhance assay sensitivity. Thus, in some embodiments, the lectin (e.g., the lectin that is in solution when the sample or sub-sample thereof is contacted with the first (and optionally the second, third, etc.) lectin) comprises a label, including an oligonucleotide, as described elsewhere herein.

[0232] In some embodiments, where the lectin is not bound to a solid substrate (e.g., a bead or array surface) when the sample or sub-sample is contacted with the lectin, the lectin bound to the target protein and / or a binding molecule that specifically binds to an epitope of the same target protein bound to the lectin (e.g., during a pre-enrichment step) can be separated or captured from the sample or sub-sample. In some embodiments, the lectin that is not bound to a solid substrate (i.e., when the sample or sub-sample is contacted with the lectin) comprises a capture moiety, e.g., one or more capture moieties described herein, such as biotin. In some embodiments, a binding molecule (e.g., a labeled binding molecule that specifically binds to an epitope of a target protein, e.g., in a step of determining the presence or level of at least one post-translationally modified target protein described herein) comprises a capture moiety, e.g., one or more capture moieties described herein, such as biotin. In some such embodiments, streptavidin bound to a solid support, such as a magnetic bead, is used to bind to the biotin on the lectin and / or binding molecule. In some embodiments, non-specifically bound material (e.g., unbound, non-target proteins) is washed away from the captured material. In some embodiments, the captured material is then dissociated from the lectin and / or biomolecule and eluted from the solid support using a salt wash or buffer. In some embodiments, the lectin and / or binding molecule is also eluted from the solid support, e.g., by disrupting the biotin-streptavidin interaction. In some embodiments, the captured lectin and / or binding molecule comprises an oligonucleotide label, and these oligonucleotide labels are amplified (e.g., using PCR primers that anneal to the oligonucleotide label) after elution from the solid support. In some embodiments, the oligonucleotide is amplified while bound to the solid support.

[0233] In some embodiments of the disclosed methods, in which the lectin is not bound to a solid substrate (e.g., a bead or an array surface) when the sample or sub-sample is contacted with the lectin, detecting the label can be performed in a manner that makes detection dependent on the proximity of the labels of the first and second lectins and / or the first and second binding molecules, e.g., a proximity ligation assay or a proximity extension assay. In some such embodiments, the detecting step comprises a proximity extension assay. In other such embodiments, the detecting step comprises a proximity ligation assay.

[0234] In other embodiments of the disclosed methods, in which the lectin is not bound to a solid substrate (e.g., a bead or an array surface) when the sample or sub-sample is contacted with the lectin, separation of the lectin bound to the target protein from other components of the sample or sub-sample comprises chromatographic separation, e.g., any of a variety of chromatographic separation methods known in the art, such as liquid chromatography, e.g., high-performance liquid chromatography (HPLC), ion-exchange chromatography, affinity chromatography, or size-exclusion chromatography. In some examples, the target protein bound to the lectin and / or binding molecule is subjected to affinity chromatography. Chromatography may be performed sequentially, in which the flow-through from one pre-enrichment is used as input for the next pre-enrichment, or in parallel, in which the protein is divided into sub-samples and pre-enriched separately, resulting in multiple pre-enriched separate fractions. In some embodiments where separation of the lectin bound to the target protein from other components of the sample or sub-sample comprises chromatographic separation, the detecting step comprises an immunoassay, e.g., an enzyme-linked immunosorbent assay, a sandwich assay, an electrochemiluminescence assay, or a multiplex immunoassay. In some such embodiments, the detecting step comprises an immunoassay performed using a binding molecule, such as an antibody, that is specific for the target protein and that is conjugated to a label, such as an oligonucleotide label as disclosed herein.

[0235] In some embodiments, at least one binding molecule of the plurality of binding molecules is conjugated to a solid support. In some embodiments, the solid support comprises beads, e.g., magnetic beads. The binding molecule to which the solid support is conjugated can be a lectin, e.g., a first lectin. This facilitates separating the first complex from other components of the sample or subsample to obtain a first pre-enriched subsample, for example, by washing away or eluting other components of the sample or subsample, resulting in their separation from the first complex. The method can then further include detecting or quantifying one or more target proteins in the first complex using, for example, an appropriate binding molecule described elsewhere herein in one or more assays, such as a proximity ligation assay or a proximity extension assay. When multiple target proteins are to be detected or quantified, the assay can be multiplexed.

[0236] In some embodiments, detection of the labels of the first and second binding molecules is used to quantify a first target protein in a sample or a subsample thereof. Any suitable technique, such as a proximity ligation assay or proximity extension assay described elsewhere herein, can be used for such quantification. In some embodiments in which a first subsample of a sample is contacted with a first lectin, the method further includes contacting an input subsample of the sample with a second plurality of binding molecules comprising a first binding molecule and a second binding molecule; and detecting the labels of the first and second binding molecules bound to the first target protein in the input subsample. The input subsample is a subsample that is not enriched for post-translationally modified target proteins and can therefore be used to determine the presence or level of one or more target proteins regardless of the presence of post-translational modifications. Detection of the labels of the first and second binding molecules bound to the first target protein in the input subsample can be used to quantify the first target protein in the input subsample. The level of the first target protein in the input sub-sample can be compared with its level in the first sub-sample to, for example, obtain an indication of the extent to which the first target protein has been modified with the first saccharide. This approach can be multiplexed, for example, each of the multiple target proteins can be detected in the first sub-sample and in the input sub-sample using a plurality of labeled binding molecules specific to each of the multiple target proteins. In some embodiments, each of the multiple target proteins is quantified in the first sub-sample and in the input sub-sample using a plurality of labeled binding molecules specific to each of the multiple target proteins. The levels of the target proteins in the input sub-sample can be compared with their levels in the first sub-sample to, for example, obtain an indication of the extent to which the target protein has been modified with the first saccharide.

[0237] B. Subject In some embodiments, the sample is obtained from a subject having cancer or a precancerous condition, an infection, transplant rejection, or other disease that directly or indirectly affects the immune system. In some embodiments, the sample is obtained from a subject suspected of having cancer or a precancerous condition, an infection, transplant rejection, or other disease that directly or indirectly affects the immune system. In some embodiments, the sample is obtained from a subject having a tumor. In some embodiments, the sample is obtained from a subject suspected of having a tumor. In some embodiments, the sample is obtained from a subject suspected of having a neoplasia. In some embodiments, the sample is obtained from a subject in remission from a tumor, cancer, or neoplasia (e.g., after chemotherapy, surgical resection, radiation, or a combination thereof). In any of the foregoing embodiments, the cancer, tumor, or neoplasia, or suspected cancer, tumor, or neoplasia, may be of the lung, colon, rectum, kidney, breast, prostate, or liver. In some embodiments, the cancer, tumor, or neoplasia, or suspected cancer, tumor, or neoplasia, is of the lung. In some embodiments, the cancer, tumor, or neoplasia, or suspected cancer, tumor, or neoplasia, is of the colon or rectum. In some embodiments, the cancer, tumor, or neoplasia, or suspected cancer, tumor, or neoplasia, is of the breast. In some embodiments, the cancer, tumor, or neoplasia, or suspected cancer, tumor, or neoplasia, is of the prostate. In any of the foregoing embodiments, the subject may be a human subject.

[0238] C.Analysis The present method can be used to diagnose the presence of a condition, particularly a cancer or precancerous condition, in a subject, characterize the condition (e.g., stage the cancer or determine the heterogeneity of the cancer), monitor the response to treatment of the condition, and predict the risk of developing the condition or the subsequent course of the condition. The present disclosure can also be useful for determining the effectiveness of a particular treatment option. A successful treatment option may result in an increase in the amount of copy number variation, rare mutation, or target protein detected in the subject's blood, since if the treatment is successful, more cancer cells will be killed and, among other things, DNA and proteins may be excreted. In other examples, this may not occur. In another example, perhaps a particular treatment option can be correlated with the profile of protein post-translational modifications and / or the genetic profile of the cancer over time. This correlation may be useful for selecting a treatment.

[0239] Additionally, if the cancer is observed to be in remission after treatment, the method can be used to monitor for residual disease or recurrence of the disease.

[0240] The types and number of cancers that can be detected include blood cancer, brain cancer, lung cancer, skin cancer, nose cancer, throat cancer, liver cancer, bone cancer, lymphoma, pancreatic cancer, skin cancer, intestinal cancer, rectal cancer, thyroid cancer, bladder cancer, kidney cancer, oral cancer, stomach cancer, solid tumors, heterogeneous tumors, homogeneous tumors, etc. The type and / or stage of cancer can be detected from genetic variations including mutations, rare mutations, indels, copy number variations, transversions, translocations, recombinations, inversions, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structural changes, gene fusions, chromosomal fusions, gene truncations, gene amplifications, gene duplications, chromosomal damage, DNA damage, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns, and abnormal changes in nucleic acid 5-methylcytosine.

[0241] In some embodiments, the methods described herein include identifying the presence of a target protein and / or DNA produced by a tumor (or neoplastic cell, or cancer cell) or by a precancerous cell.

[0242] Genetic data can also be used to characterize specific forms of cancer. Cancers are often heterogeneous in both composition and stage classification. Genetic profile data can enable characterization of specific subtypes of cancer, which may be important in the diagnosis or treatment of specific subtypes. This information can also provide subjects or practitioners with clues regarding the prognosis of specific types of cancer, allowing either subjects or practitioners to adapt treatment options to the progression of the disease. Some cancers can progress to become more aggressive and genetically unstable. Other cancers can remain benign, inactive, or dormant. The systems and methods of the present disclosure can be useful in determining disease progression.

[0243] Furthermore, the methods of the present disclosure can be used to characterize the heterogeneity of abnormal conditions in a subject. Such methods can include, for example, generating a target protein profile from a subject, where the profile includes multiple data obtained as a result of the detection described herein, optionally in combination with additional data, such as epigenetic changes, copy number variations, and / or mutations. In some embodiments, the abnormal condition is cancer or a precancerous condition. In some embodiments, the abnormal condition can result in a heterogeneous genomic population. In the example of cancer, it has been found that some tumors contain tumor cells at different stages of cancer. In other examples, the heterogeneity can include multiple foci of disease. Again, in the example of cancer, there can be multiple tumor foci, and perhaps one or more foci are the result of metastasis that has spread from the primary site.

[0244] The method can be used to generate a profile, fingerprint, or data set that is a compilation of information from different cells in a heterogeneous disease, which may include target protein post-translational modifications, identities, levels, copy number variation, epigenetic diversity, or other mutation analyses, alone or in combination.

[0245] This method can be used to diagnose, prognose, monitor or observe cancer or other diseases.In some embodiments, the method herein does not involve diagnosing, prognosing or monitoring fetus, and therefore is not intended for non-invasive prenatal testing.In other embodiments, these methodologies can be used in pregnant subjects to diagnose, prognose, monitor or observe cancer or other diseases in unborn subjects, in which DNA and other polynucleotides may co-circulate with maternal molecules.

[0246] D. DNA analysis; sample partitioning into multiple subsamples In some embodiments described herein, the disclosed method further comprises analyzing DNA in a sample (which may be a separate sample from the same subject or the same sample). For example, analyzing DNA, e.g., cell-free DNA, in combination with analyzing post-translationally modified proteins can improve the specificity and / or sensitivity of a method for detecting an abnormal condition, such as the presence of a disease. In such a method, different forms of DNA (e.g., hypermethylated DNA and hypomethylated DNA) can be physically separated based on one or more characteristics of the DNA. This approach can be used, for example, to determine whether a particular sequence is hypermethylated or hypomethylated. Detecting abnormal levels of one or more post-translationally modified proteins in conjunction with detecting abnormal DNA features (whether sequence-based, epigenetic, or both) can provide greater specificity and / or sensitivity for identifying abnormal conditions than detecting DNA features alone or the levels of one or more post-translationally modified proteins alone.

[0247] Methylation profiling can involve determining the methylation pattern across different regions of a genome. For example, molecules can be divided based on the degree of methylation (e.g., the relative number of methylated nucleic acid bases per molecule), and then sequenced. The sequences of molecules within different divisions can then be mapped to a reference genome. This can reveal regions of the genome that are more highly methylated or less highly methylated than other regions. In this way, genomic regions, as opposed to individual molecules, can have different degrees of methylation.

[0248] Partitioning nucleic acid molecules in a sample can increase rare signal, for example, by enriching rare nucleic acid molecules that are more commonly found in one partition of the sample. For example, genetic variation present in hypermethylated DNA but less (or not at all) present in hypomethylated DNA can be more easily detected by partitioning the sample into hypermethylated and hypomethylated nucleic acid molecules. By analyzing multiple partitions of a sample, multidimensional analysis of single molecules can be performed, thus achieving greater sensitivity. Partitioning can include physically dividing nucleic acid molecules into partitions or subsamples based on the presence or absence of one or more methylated nucleic acid bases. Samples can be divided into partitions or subsamples based on features indicative of differential gene expression or pathology. Samples can be divided based on features or combinations that result in signal differences between normal and disease states during the analysis of nucleic acids, such as cell-free DNA (cfDNA), non-cfDNA, tumor DNA, circulating tumor DNA (ctDNA), and cell-free nucleic acid (cfNA).

[0249] In some embodiments, hypermethylated and / or hypomethylated variable epigenetic target regions are analyzed to determine whether they exhibit differential methylation characteristic of tumor cells, or cell types that do not normally contribute to the DNA sample (e.g., cfDNA) being analyzed, and / or cells of specific immune cell types.

[0250] In some cases, the heterogeneous DNA in a sample is divided into two or more partitions (for example, at least three, four, five, six or seven partitions). In some embodiments, each partition is differentially tagged. Then, the tagged partitions can be pooled together for collective sample preparation and / or sequencing. The dividing-tagging-pooling step can be performed more than once, and each round of dividing step is performed based on different characteristics (examples provided herein) and tagged using differential tags that are distinct from other divisions and dividing means. In other cases, the differentially tagged partitions are sequenced separately.

[0251] In some embodiments, sequence reads obtained from differentially tagged and pooled DNA are analyzed in silico. Tags are used to sort reads from different distributions. Analysis to detect genetic variants can be performed at the distribution level as well as at the overall nucleic acid population level. For example, analysis can include in silico analysis to determine genetic variants, such as CNVs, SNVs, indels, and fusions, in the nucleic acids within each distribution. In some cases, in silico analysis can include determining chromatin structure. For example, the coverage of sequence reads can be used to determine nucleosome positioning in chromatin. Higher coverage can correlate with higher nucleosome occupancy within a genomic region, while lower coverage can correlate with lower nucleosome occupancy or nucleosome-depleted regions (NDRs).

[0252] Examples of characteristics that can be used in the partitioning step include sequence length, methylation level, nucleosome binding, sequence mismatch, immunoprecipitation, and / or proteins binding to DNA. The resulting partitions may include one or more of the following nucleic acid forms: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), shorter DNA fragments, and longer DNA fragments. In some embodiments, a partitioning step based on cytosine modification (e.g., cytosine methylation) or methylation is generally performed, optionally combined with at least one additional partitioning step that may be based on any of the above DNA characteristics or forms. In some embodiments, a heterogeneous population of nucleic acids is partitioned into nucleic acids with one or more epigenetic modifications and nucleic acids without one or more epigenetic modifications. Examples of epigenetic modifications include the presence or absence of methylation; the level of methylation; the type of methylation (e.g., 5-methylcytosine or other types of methylation, such as adenine methylation and / or cytosine hydroxymethylation); and the association and level of association with one or more proteins, such as histones. Alternatively or additionally, the heterogeneous population of nucleic acids can be divided into nucleic acid molecules with nucleosomes and nucleic acid molecules lacking nucleosomes. Alternatively or additionally, the heterogeneous population of nucleic acids can be divided into single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Alternatively or additionally, the heterogeneous population of nucleic acids can be divided based on the length of the nucleic acid (e.g., molecules up to 160 bp and molecules longer than 160 bp).

[0253] The agent used to partition the population of nucleic acids within a sample can be an affinity agent, e.g., an antibody with desired specificity, a natural binding partner or variant thereof (Bock et al., Nat Biotech 28: 1106-1114 (2010); Song et al., Nat Biotech 29: 68-72 (2011)), or an artificial peptide selected, e.g., by phage display, to have specificity for a given target. In some embodiments, the agent used in the partitioning step is an agent that recognizes a modified nucleobase. In some embodiments, the modified nucleobase recognized by the agent is a modified cytosine, such as methylcytosine (e.g., 5-methylcytosine). In some embodiments, the modified nucleobase recognized by the agent is the product of a procedure that affects a first nucleobase in the DNA of the sample differently from a second nucleobase in the DNA. In some embodiments, the modified nucleobase can be a "converted nucleobase," i.e., one whose base-pairing specificity has been altered by the procedure. For example, certain procedures convert unmethylated or unmodified cytosine to dihydrouracil, or more commonly, at least one modified or unmodified form of cytosine undergoes deamination, thereby generating uracil (considered a modified nucleobase in the context of DNA) or a further modified form of uracil. Examples of partitioning agents include antibodies, for example, antibodies that recognize modified nucleobases, which may be modified cytosines, such as methylcytosine (e.g., 5-methylcytosine). In some embodiments, the partitioning agent is an antibody that recognizes modified cytosines other than 5-methylcytosine, such as 5-carboxylcytosine (5caC). Other partitioning agents include the methyl-binding domains (MBDs) and methyl-binding proteins (MBPs) described herein, including proteins such as MeCP2.

[0254] Additional non-limiting examples of partitioning agents are histone-binding proteins that can separate histone-bound nucleic acids from free or unbound nucleic acids. Examples of histone-binding proteins that can be used in the methods disclosed herein include RBBP4, RbAp48, and SANT domain peptides.

[0255] In some embodiments, the partitioning step can include both binary partitioning and partitioning based on the degree / level of modification. For example, methylated fragments can be partitioned by methylated DNA immunoprecipitation (MeDIP), or all methylated fragments can be partitioned from unmethylated fragments using a methyl-binding domain protein (e.g., MethylMiner Methylated DNA Enrichment Kit (ThermoFisher Scientific)). An additional partitioning step can then involve eluting fragments with different levels of methylation by adjusting the salt concentration in the solution containing the methyl-binding domain and bound fragments. As the salt concentration increases, more highly methylated fragments elute.

[0256] In some cases, the final partition is enriched for nucleic acids with different degrees of modification (overrepresentative or underrepresentative of the modification). Overrepresentation and underrepresentation can be defined by the number of modifications carried by a nucleic acid compared to the median number of modifications per strand in the population. For example, if the median number of 5-methylcytosine residues in nucleic acids in a sample is 2, nucleic acids containing more than two 5-methylcytosine residues are overrepresented for this modification, and nucleic acids with one or no 5-methylcytosine residues are underrepresented. The effect of affinity separation is to enrich nucleic acids with a high abundance of the modification in the bound phase and nucleic acids with a low abundance of the modification in the non-bound phase (i.e., in solution). The nucleic acids in the bound phase can be eluted and then processed.

[0257] When using MeDIP or the MethylMiner® Methylated DNA Enrichment Kit (ThermoFisher Scientific), sequential elution can be used to separate different levels of methylation. For example, the nucleic acid population can be contacted with the MBD from the kit attached to magnetic beads to separate the low-methylated (unmethylated) distribution from the methylated distribution. The beads are used to separate the methylated nucleic acids from the unmethylated nucleic acids. One or more sequential elution steps are then performed to elute nucleic acids with different levels of methylation. For example, the first set of methylated nucleic acids can be eluted at a salt concentration of 160 mM or higher, e.g., at least 150 mM, at least 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, or 2000 mM. After eluting such methylated nucleic acids, magnetic separation is again used to separate nucleic acids with higher levels of methylation from nucleic acids with lower levels of methylation. The elution and magnetic separation steps can be repeated to generate various partitions, such as a low-methylated partition (enriched for nucleic acids with no methylation), a methylated partition (enriched for nucleic acids with low levels of methylation), and a high-methylated partition (enriched for nucleic acids with high levels of methylation).

[0258] In some methods, nucleic acids bound to an agent used for affinity separation-based partitioning are subjected to a wash step, which washes away nucleic acids that are weakly bound to the affinity agent. Such nucleic acids may be enriched for nucleic acids that are modified to a degree close to the mean or median (i.e., intermediate between the nucleic acids that remain bound to the solid phase and the nucleic acids that are not bound to the solid phase upon initial contact of the sample with the agent).

[0259] Affinity separation results in at least two, and sometimes three or more, partitions of nucleic acids with different degrees of modification. Although the partitions are still separate, the nucleic acids of at least one partition, and usually two or three (or more) partitions, are linked to nucleic acid tags, usually provided as components of adapters, and nucleic acids in different partitions receive different tags that distinguish members of one partition from members of another partition. Tags linked to nucleic acid molecules of the same partition can be the same or different from each other. However, if different from each other, the tags may share part of their code so that the molecules to which they are bound can be identified as belonging to a specific partition.

[0260] For further details regarding partitioning nucleic acid samples based on characteristics such as methylation, see WO2018 / 119452, which is incorporated herein by reference.

[0261] In some embodiments, the partitioning step is carried out by contacting the nucleic acid with a methyl-binding domain ("MBD") of a methyl-binding protein ("MBP"). In some such embodiments, the nucleic acid is contacted with the entire MBP. In some embodiments, the MBD binds to 5-methylcytosine (5mC), and the MBP comprises the MBD, referred to interchangeably herein as a methyl-binding protein or a methyl-binding domain protein. In some embodiments, the MBD is coupled to paramagnetic beads, e.g., Dynabeads® M-280 streptavidin, via a biotin linker. Partitioning into fractions with different degrees of methylation can be carried out by eluting the fractions with increasing NaCl concentrations.

[0262] In some embodiments, the bound DNA is eluted by contacting the antibody or MBD with a protease, such as proteinase K. This can be performed instead of or in addition to the elution step using NaCl discussed above.

[0263] Examples of modified nucleobase-recognizing agents contemplated herein include, but are not limited to, the following: (a) MeCP2 is a protein that preferentially binds 5-methyl-cytosine over unmodified cytosine. (b) RPL26, PRP8 and the DNA mismatch repair protein MHS6 bind preferentially to 5-hydroxymethyl-cytosine over unmodified cytosine. (c) FOXK1, FOXK2, FOXP1, FOXP4, and FOXI3 bind 5-formyl-cytosine more preferably than unmodified cytosine (Iurlaro et al., Genome Biol. 14: R119 (2013)). (d) An antibody specific for one or more methylated or modified nucleobases or their conversion products, e.g., 5mC, 5caC, or DHU.

[0264] Generally, elution is a function of the number of modifications (e.g., the number of methylation sites) per molecule, with molecules with more methylation eluting at higher salt concentrations. A series of elution buffers with increasing NaCl concentrations can be used to elute DNA into distinct populations based on the degree of methylation. Salt concentrations can range from about 100 mM to about 2500 mM NaCl. In one embodiment, the process results in three partitions. The molecules are contacted with a solution at a first salt concentration, which contains an agent that recognizes modified nucleobases and contains molecules that can bind to a capture moiety, such as streptavidin. At the first salt concentration, some populations of molecules bind to the agent, while others remain unbound. The unbound population can be separated as a "hypomethylated" population. For example, the first partition, enriched for hypomethylated forms of DNA, remains unbound at low salt concentrations, e.g., 100 mM or 160 mM. A second partition enriched in moderately methylated DNA is eluted using an intermediate salt concentration, e.g., between 100 mM and 2000 mM, and similarly separated from the sample. A third partition enriched in highly methylated forms of DNA is eluted using a high salt concentration, e.g., at least about 2000 mM.

[0265] In some embodiments, methylated DNA is purified using a monoclonal antibody raised against 5-methylcytidine (5mC). To obtain single-stranded DNA fragments, the DNA is denatured, for example, at 95°C. The DNA bound to the antibody is immunoprecipitated using protein G bound to standard or magnetic beads and washed after incubation with the anti-5mC antibody. The DNA can then be eluted. The partition may include unprecipitated DNA and one or more partitions eluted from the beads.

[0266] In some embodiments, the DNA fraction is desalted and concentrated in preparation for the enzymatic steps of library preparation.

[0267] E. Adapter ligation or addition; tagging In some embodiments, the disclosed methods further include analyzing DNA in a sample (which may be a different sample from the same subject or the same sample). In such methods, adapters can be added to the DNA. This can be done in parallel with the amplification procedure, for example, by providing adapters to the 5' portion of the primers (when PCR is used, this can be referred to as library preparation-PCR or LP-PCR). In some embodiments, the adapters are added by other techniques, such as ligation. In some such methods, before the partitioning step or the capturing step, a first adapter is added to the nucleic acid by ligation to its 3' end, which can include ligation to single-stranded DNA. The adapter can be used, for example, as a priming site for second-strand synthesis using a universal primer and DNA polymerase. A second adapter can then be ligated to at least the 3' end of the second strand of the now double-stranded molecule. In some embodiments, the first adaptor comprises an affinity tag such as biotin, and the nucleic acid ligated to the first adaptor is bound to a solid support (e.g., beads) that may comprise a binding partner of the affinity tag, such as streptavidin. For further discussion of related procedures, see Gansauge et al., Nature Protocols 8: 737-748 (2013). Commercially available kits for sequencing library preparations that are compatible with single-stranded nucleic acids are available, such as the Accel-NGS® Methyl-Seq DNA Library Kit from Swift Biosciences. In some embodiments, after adaptor ligation, the nucleic acid is amplified.

[0268] Preferably, the adapters contain a sufficient number of different tags such that the number of tag combinations results in a low probability, e.g., 95%, 99%, or 99.9%, that two nucleic acids with the same start and end points will receive the same tag combination. Adapters, whether they have the same or different tags, can contain the same or different primer binding sites, although preferably the adapters contain the same primer binding sites.

[0269] In some embodiments, after adapter binding, the nucleic acid is subjected to amplification, which can be, for example, using universal primers that recognize primer-binding sites in the adapters.

[0270] In some embodiments, after adapter binding, the DNA is partitioned, which involves contacting the DNA with an agent that preferentially binds to nucleic acids with epigenetic modifications. The nucleic acids are partitioned into at least two subsamples that differ in the degree to which the nucleic acids have the modification from binding to the agent. For example, if the agent has affinity for nucleic acids with the modification, nucleic acids that are overrepresented in the modification (compared to the median abundance of the population) will preferentially bind to the agent, while nucleic acids that are underrepresented in the modification will not bind to the agent or will be more easily eluted from the agent. Thus, nucleic acids can be amplified from primers that bind to the primer-binding sites in the adapter. Alternatively, partitioning can be performed before adapter binding, in which case the adapter can include a discriminatory tag containing a component that identifies which partition the molecule was present in.

[0271] In some embodiments, the nucleic acid is ligated at both ends to a Y-shaped adapter comprising a primer binding site and a tag, and the molecule is amplified.

[0272] Tagging of DNA molecules is the procedure of attaching or associating a tag to a DNA molecule. Such a tag can be a molecule, such as a nucleic acid, that contains information that indicates the characteristics of the molecule to which the tag is associated. For example, a molecule can have a sample tag (that distinguishes molecules in one sample from molecules in a different sample) or a molecular tag / molecular barcode / barcode (that distinguishes different molecules from each other (in both unique and non-unique tagging scenarios)). For methods that involve a partitioning step, a partitioning tag (that distinguishes molecules in one partition from molecules in a different partition) can be included. In some embodiments, the adapter added to the DNA molecule comprises a tag. In certain such embodiments, the tag can comprise a barcode or a combination of barcodes. As used herein, the term "barcode" can refer to a nucleic acid molecule having a specific nucleotide sequence, or to a molecule that identifies the nucleotide sequence itself, depending on the context. A barcode can have, for example, between 10 and 100 nucleotides. A group of barcodes can have a degenerate sequence or a sequence with a certain Hamming distance that is desirable for a particular purpose. Thus, for example, a molecular barcode can be composed of one barcode or a combination of two barcodes, each attached to a different end of the molecule. Additionally or alternatively, different sets of molecular barcodes or molecular tags can be used for different distributions and / or samples, such that the barcodes function as molecular tags through their individual sequences and also identify the distributions and / or samples to which they correspond based on the set to which they belong.

[0273] In some embodiments, two or more partitions, for example, each partition, are differentially tagged. To associate a tag (or multiple tags) with a specific partition, tags can be used to label individual polynucleotide population partitions. Alternatively, tags can be used in embodiments that do not use a partitioning step. In some embodiments, a single tag can be used to label a specific partition. In some embodiments, multiple different tags can be used to label a specific partition. In embodiments that use multiple different tags to label specific partitions, the set of tags used to label one partition can be easily distinguished from the set of tags used to label other partitions. In some embodiments, tags may have additional functions, for example, tags may be used to index sample sources, or may be used as unique molecular identifiers (e.g., to improve sequencing data quality by distinguishing sequencing errors from mutations, as in Kinde et al., Proc Nat'l Acad Sci USA 108: 9530-9535 (2011); Kou et al., PLoS ONE, 11: e0146638 (2016)), or may be used as non-unique molecular identifiers, as described, for example, in U.S. Pat. No. 9,598,731. Similarly, in some embodiments, tags may have additional functions, for example, tags may be used to index sample sources, or may be used as non-unique molecular identifiers (e.g., to improve sequencing data quality by distinguishing sequencing errors from mutations).

[0274] In some embodiments, distribution tagging involves tagging molecules in each distribution with a distribution tag. After recombining the distributions (e.g., to reduce the number of required sequencing runs and avoid unnecessary costs) and sequencing the molecules, the distribution tag identifies the original distribution. In another embodiment, different distributions are tagged with different sets of molecular tags, e.g., consisting of pairs of barcodes. In this way, each molecular barcode not only indicates the original distribution, but also serves to distinguish molecules within the distribution. For example, a first set of 35 barcodes can be used to tag molecules in a first distribution, while a second set of 35 barcodes can be used to tag molecules in a second distribution.

[0275] In some embodiments, after partitioning and tagging with partition tags, the molecules can be pooled for sequencing in a single run. In some embodiments, sample tags are added to molecules, for example, in a step after partition tag addition and pooling. Sample tags can facilitate pooling materials generated from multiple samples for sequencing in a single sequencing run.

[0276] Alternatively, in some embodiments, distribution tags can be associated with samples and distributions. As a simple example, a first tag can indicate a first distribution of a first sample, a second tag can indicate a second distribution of the first sample, a third tag can indicate a first distribution of a second sample, and a fourth tag can indicate a second distribution of the second sample.

[0277] Tags can be attached to molecules that have already been distributed based on one or more characteristics, but the final tagged molecules in the library may no longer have those characteristics.For example, single-stranded DNA molecules can be distributed and tagged, but the final tagged molecules in the library may be double-stranded.Similarly, DNA can be distributed based on different levels of methylation, but in the final library, the tagged molecules derived from these molecules may be unmethylated.Therefore, the tags attached to molecules in the library generally represent the characteristics of the "parent molecule" from which the final tagged molecules are derived, but do not necessarily represent the characteristics of the tagged molecules themselves.

[0278] As an example, barcodes 1, 2, 3, 4, etc. are used to tag and label molecules in a first distribution, barcodes A, B, C, D, etc. are used to tag and label molecules in a second distribution, and barcodes a, b, c, d, etc. are used to tag and label molecules in a third distribution. The differentially tagged distributions can be pooled before sequencing. The differentially tagged distributions can be sequenced separately or can be sequenced together, for example, in the same flow cell of an Illumina sequencer.

[0279] After sequencing, analysis of reads can be performed at the level of each distribution and at the level of the whole DNA population. Tags are used to sort reads from different distributions. Analysis can include in silico analysis to determine genetic and epigenetic diversity (one or more of methylation, chromatin structure, etc.) using sequence information, genome coordinate length, coverage, and / or copy number. In some embodiments, higher coverage can be correlated with higher nucleosome occupancy within a genomic region, while lower coverage can be correlated with lower nucleosome occupancy or nucleosome-depleted regions (NDRs).

[0280] Molecular tagging refers to tagging practices that enable the identification of DNA molecules from which sequence reads originate. Tagging strategies can be divided into unique tagging and non-unique tagging. In unique tagging, all or substantially all of the molecules in a sample have different tags, and thus reads can be assigned to the original molecule based solely on tag information. Tags used in such methods are sometimes referred to as "unique tags." In non-unique tagging, different molecules in the same sample may have the same tag, and therefore other information is used in addition to tag information to assign sequence reads to the original molecule. Such information may include start and end coordinates, coordinates to which the molecule is mapped, start coordinates alone, or end coordinates alone. Tags used in such methods are sometimes referred to as "non-unique tags." Therefore, it is not necessary to uniquely tag every molecule in a sample. It is sufficient to uniquely tag molecules that fall within an identifiable class within a sample. Thus, molecules within different identifiable families can have the same tag without losing information about the identity of the tagged molecule.

[0281] In certain embodiments of non-unique tagging, the number of different tags used may be sufficient so that there is a very high probability (e.g., at least 99%, at least 99.9%, at least 99.99%, or at least 99.999%) that all DNA molecules in a particular group have different tags. Note that when barcodes are used as tags, and when barcodes are attached to both ends of a molecule, e.g., randomly, a combination of barcodes may together constitute a tag. This number is in turn a function of the number of molecules included in the call. For example, a class may be all molecules that map to the same start-end position on a reference genome. A class may be all molecules that map to a particular locus, e.g., a particular base or a particular region (e.g., up to 100 bases or a gene or exon of a gene). In certain embodiments, the number of different tags used to uniquely identify the number of molecules in a class, z, is 2.* z, 3 * z, 4 * z, 5 * z, 6 * z, 7 * z, 8 * z, 9 * z, 10 * z, 11 * z, 12 * z, 13 * z, 14 * z, 15 * z, 16 * z, 17 * z, 18 * z, 19 * z, 20 * z or 100 * z (e.g., lower limit) ~ 100,000 * z, 10,000 * z, 1000 * z or 100 * z (e.g., upper limit).

[0282] For example, in a sample of about 5 ng to 30 ng of cell-free DNA, approximately 3,000 molecules are expected to map to a particular nucleotide coordinate, with approximately 3 to 10 molecules with any given start coordinate expected to share the same end coordinate. Therefore, approximately 50 to 50,000 different tags (e.g., between about 6 and 220 barcode combinations) may be sufficient to uniquely tag all such molecules. To uniquely tag all 3,000 molecules mapped across nucleotide coordinates, approximately 1 million to 20 million different tags would be required.

[0283] Generally, assignment of unique or non-unique tag barcodes in reactions follows the methods and systems described in U.S. Patent Applications 20010053519, 20030152490, 20110160078, and U.S. Patent Nos. 6,582,908, 7,537,898, and 9,598,731. Tags can be randomly or non-randomly linked to sample nucleic acids.

[0284] In some embodiments, the tagged nucleic acids are sequenced after loading into a microwell plate. The microwell plate may have 96, 384, or 1536 microwells. In some cases, the tagged nucleic acids are introduced into the microwells at a predicted ratio of unique tags. For example, unique tags can be loaded such that more than about 1, more than about 2, more than about 3, more than about 4, more than about 5, more than about 6, more than about 7, more than about 8, more than about 9, more than about 10, more than about 20, more than about 50, more than about 100, more than about 500, more than about 1000, more than about 5000, more than about 10000, more than about 50,000, more than about 100,000, more than about 500,000, more than about 1,000,000, more than about 10,000,000, more than about 50,000,000, or more than about 1,000,000,000 unique tags are loaded per genomic sample. In some cases, the unique tags can be loaded such that less than about 2, less than about 3, less than about 4, less than about 5, less than about 6, less than about 7, less than about 8, less than about 9, less than about 10, less than about 20, less than about 50, less than about 100, less than about 500, less than about 1000, less than about 5000, less than about 10000, less than about 50,000, less than about 100,000, less than about 500,000, less than about 1,000,000, less than about 10,000,000, less than about 50,000,000, or less than about 1,000,000,000 unique tags are loaded per genomic sample.In some cases, the average number of unique tags loaded per sample genome is less than about 1, less than about 2, less than about 3, less than about 4, less than about 5, less than about 6, less than about 7, less than about 8, less than about 9, less than about 10, less than about 20, less than about 50, less than about 100, less than about 500, less than about 1000, less than about 5000, less than about 10000, less than about 50,000, less than about 100,000, less than about 500,000, less than about 1,000,000, less than about 10,000,000, less than about 50,000,000, or less than about 1,000,000,000, or more than about 1, more than 2, or more than about 1, more than 2, or more than about 1, more than 2, or more than about 10,000,000, or more than about 2 ... more than about 3, more than about 4, more than about 5, more than about 6, more than about 7, more than about 8, more than about 9, more than about 10, more than about 20, more than about 50, more than about 100, more than about 500, more than about 1000, more than about 5000, more than about 10000, more than about 50,000, more than about 100,000, more than about 500,000, more than about 1,000,000, more than about 10,000,000, more than about 500,000, more than about 1,000,000, more than about 10,000,000, more than about 50,000,000, or more than about 1,000,000,000 unique tags.

[0285] A preferred format uses 20-50 different tags (e.g., barcodes) ligated to both ends of a target nucleic acid. For example, 35 different tags (e.g., barcodes) ligated to both ends of a target molecule generate 35 x 35 permutations, which is equivalent to 1225 for 35 tags. This number of tags is sufficient so that different molecules with the same start and end points have a high probability (e.g., at least 94%, 99.5%, 99.99%, 99.999%) of receiving different tag combinations. Other barcode combinations include any number between 10 and 500, such as about 15 x 15, about 35 x 35, about 75 x 75, about 100 x 100, about 250 x 250, and about 500 x 500.

[0286] In some cases, the unique tag may be an oligonucleotide of predetermined, random, or semi-random sequence. In other cases, multiple barcodes may be used, and therefore the barcodes are not necessarily unique to each other within the plurality. In this example, the barcode may be ligated to an individual molecule, and thus the combination of the barcode and the sequence to which it may be ligated generates a unique sequence that can be tracked individually. As described herein, detection of a non-unique barcode in combination with sequence data at the beginning (start) and end (end) of a sequence read may allow assignment of a unique identity to a particular molecule. The length or number of base pairs of an individual sequence read may also be used to assign a unique identity to such a molecule. As described herein, a unique identity may be assigned to a fragment from a single strand of nucleic acid, thereby allowing subsequent identification of the fragment from the parent strand.

[0287] F. Enrichment / Capture Step; Amplification The method disclosed herein can include enriching, capturing or isolating post-translationally modified proteins and / or target proteins, and / or enriching, capturing or isolating DNA, such as cfDNA target regions.In some embodiments, capturing includes contacting post-translationally modified proteins and / or target proteins with binding molecules specific for PTMs and / or target proteins, and / or contacting DNA with probes specific for target regions.Enrichment or capture can be carried out on any sample or sub-sample described herein using any suitable method known in the art.

[0288] In some embodiments, binding molecules specific for PTMs or target proteins, or probes specific for DNA target regions, contain a capture moiety that facilitates enrichment or capture of DNA hybridized to the target protein or probe, respectively. In some embodiments, the capture moiety is biotin. In some such embodiments, streptavidin bound to a solid support, such as magnetic beads, is used to bind to the biotin. In some embodiments, non-specifically bound material (e.g., DNA not containing the target region) is washed away from the captured material. In some embodiments, the captured material is then dissociated from the probe and eluted from the solid support using a buffer containing a salt wash or another DNA denaturing agent. In some embodiments, the binding molecule and / or probe is also eluted from the solid support, e.g., by disrupting the biotin-streptavidin interaction. In some embodiments, the captured DNA and / or oligonucleotide label is amplified after elution from the solid support. In some such embodiments, DNA containing an adapter is amplified using PCR primers that anneal to the adapter. In some embodiments, the captured DNA is amplified while still bound to the solid support. In some such embodiments, amplification involves the use of PCR primers that anneal to a sequence within the adapter and a PCR primer that anneals to a sequence within the probe that anneals to the target region of DNA.

[0289] In some embodiments, the methods herein include enriching or capturing DNA containing epigenetic and / or sequence-variable target regions. Such regions can be captured from an aliquot of a sample (e.g., a sample that has undergone adapter binding and amplification), while distributing the DNA with an agent that recognizes methylcytosine is performed on another aliquot of the sample. Enriching or capturing DNA containing epigenetic and / or sequence-variable target regions can include contacting the DNA with a first or second set of target-specific probes. Such target-specific probes can have any of the features described herein, including, but not limited to, the embodiments above and the probe section below, with respect to the set of target-specific probes. The capturing step can be performed on one or more sub-samples prepared during the methods disclosed herein. In some embodiments, DNA is captured from a first sub-sample or a second sub-sample, e.g., a first sub-sample and a second sub-sample. In some embodiments, the sub-samples are differentially tagged (e.g., as described herein) and then pooled before undergoing capture. Exemplary methods for capturing DNA containing epigenetic target regions and / or sequence variable target regions can be found, for example, in WO2020 / 160414, which is hereby incorporated by reference herein.

[0290] The capturing step can be carried out using the conditions suitable for specific nucleic acid hybridization, and the conditions generally depend to some extent on the characteristics of the probe, such as length, base composition, etc. Suitable conditions are well known to those skilled in the art, taking into account the general knowledge in the art of nucleic acid hybridization. In some embodiments, a complex of target-specific probe and DNA is formed.

[0291] In some embodiments, the methods described herein include capturing multiple sets of target regions of cfDNA obtained from a subject. The target regions may vary depending on whether they originate from a tumor, a healthy cell, or a specific cell type. The capturing step results in a captured set of cfDNA molecules. In some embodiments, cfDNA molecules corresponding to the set of sequence-variable target regions are captured with a higher capture yield in the captured set of cfDNA molecules than cfDNA molecules corresponding to the set of epigenetic target regions. In some embodiments, the methods described herein include contacting cfDNA obtained from a subject with a set of target-specific probes, wherein the set of target-specific probes is configured to capture cfDNA corresponding to the set of sequence-variable target regions with a higher capture yield than cfDNA corresponding to the set of epigenetic target regions. For additional discussions regarding capturing, capture yield, and related aspects, see WO2020 / 160414, which is incorporated herein by reference for all purposes.

[0292] Because analyzing sequence variable target regions with sufficient reliability or accuracy may require a deeper sequencing depth than that required for analyzing epigenetic target regions, it may be beneficial to capture the cfDNA corresponding to sequence variable target region set with a higher capture yield than the cfDNA corresponding to epigenetic target region set.The amount of data required to determine fragmentation patterns (for example, to test the disruption of transcription start sites or CTCF binding sites) or to determine fragment abundance (for example, in hypermethylation distribution and hypomethylation distribution) is generally less than the amount of data required to determine the presence or absence of cancer-related sequence mutations.By capturing target region sets with different yields, it can be easier to sequence target regions to different sequencing depths in the same sequencing run (for example, by using pooled mixtures and / or in the same sequencing cell).

[0293] In some embodiments, the DNA is amplified. In some embodiments, the amplification is performed before the capturing step. In some embodiments, the amplification is performed after the capturing step. In some embodiments, the amplification is performed before and after the capturing step. In various embodiments, the method further comprises sequencing the captured DNA, for example, to different sequencing depths for the set of epigenetic target regions and the set of sequence variable target regions, consistent with the discussion herein.

[0294] In some embodiments, the capturing step is performed using probes for a set of sequence variable target regions and probes for a set of epigenetic target regions simultaneously in the same vessel, e.g., the probes for the set of sequence variable target regions and the set of epigenetic target regions are in the same composition. This approach results in a relatively streamlined workflow.

[0295] In some embodiments, an adapter is included in the DNA described herein. In some embodiments, a tag is included in the DNA, which may be or include a barcode. In some embodiments, such a tag is included in the adapter. The tag can facilitate identification of the origin of the nucleic acid. For example, a barcode can be used to enable identification of the source (e.g., subject) from which the DNA originated after pooling multiple samples for parallel sequencing. This can be done in parallel with the amplification procedure, for example, by providing the barcode in the 5' portion of a primer, for example, as described herein. In some embodiments, the adapter and tag / barcode are provided by the same primer or primer set. For example, the barcode can be located 3' of the adapter and 5' of the portion of the primer that hybridizes to the target. Alternatively, the barcode can be added by other techniques, such as ligation, optionally together with the adapter in the same ligation substrate.

[0296] Additional details regarding amplification, tags and barcodes are discussed herein, which may be combined with any of these embodiments to the extent practicable.

[0297] G. Captured set; target area In some embodiments, the nucleic acids captured or enriched using the methods described herein include captured DNA, for example, one or more captured sets of DNA. In some embodiments, the captured DNA includes a target region that is differentially methylated in different immune cell types. In some embodiments, the immune cell types include rare or closely related immune cell types, for example, activated and naive lymphocytes or myeloid cells at different differentiation stages.

[0298] In some embodiments, the set of captured epigenetic target regions captured from a sample or a first sub-sample comprises a hypermethylated variable target region. In some embodiments, the hypermethylated variable target region is differentially or exclusively hypermethylated in one cell type, or one immune cell type, or one immune cell type in a cluster. In some embodiments, the hypermethylated variable target region is hypermethylated to a degree that it is distinctly more highly or exclusively present in one cell type, or one immune cell type, or one immune cell type in a cluster. Such a hypermethylated variable target region may be hypermethylated in other cell types, but not as much as observed in one cell type. In some embodiments, the hypermethylated variable target region shows lower methylation in healthy cfDNA than in at least one other tissue type.

[0299] In some embodiments, the epigenetic target region set captured from sample or second sub-sample comprises hypomethylated variable target region.In some embodiments, hypomethylated variable target region is exclusively hypomethylated in one cell type, or in one immune cell type, or in one immune cell type in a cluster.In some embodiments, hypomethylated variable target region is hypomethylated to the extent that it is exclusively present in one cell type or one immune cell type, or in one immune cell type in a cluster.Such hypomethylated variable target region can be hypomethylated in other cell types, but not to the extent that it is observed in one cell type.In some embodiments, hypomethylated variable target region shows higher methylation in at least one other tissue type in healthy cfDNA.

[0300] Without being bound by any particular theory, in individuals with cancer, proliferating or activated immune cells and / or cancer cells may excrete more DNA into the bloodstream than immune cells in healthy individuals and / or than healthy cells of the same tissue type. Therefore, the distribution of cell types and / or tissues of origin of cfDNA may change during carcinogenesis. Thus, variations in hypermethylation and / or hypomethylation may be indicative of disease. For example, elevated levels of hypermethylated and / or hypomethylated variable target regions in the subsample after the distribution step may be indicative of the presence (or recurrence, depending on the subject's medical history) of cancer.

[0301] Exemplary hypermethylated and hypomethylated variable target regions useful for distinguishing various cell types, including, but not limited to, immune cell types, have been identified by analyzing DNA from various cell types by whole-genome bisulfite sequencing, as described, for example, in Scott, CA, Duryea, JD, MacKay, H. et al., "Identification of cell type-specific methylation signals in bulk whole genome bisulfite sequencing data," Genome Biol 21, 156 (2020) (doi.org / 10.1186 / s13059-020-02065-5). Whole-genome bisulfite sequencing data is available from the Blueprint consortium, available online at dcc.blueprint-epigenome.eu.

[0302] In some embodiments, the first and second captured target region sets comprise DNA corresponding to a sequence variable target region set and DNA corresponding to an epigenetic target region set, respectively, as described, for example, in WO2020 / 160414. The first captured set and the second captured set can be combined to obtain a combined captured set. The sequence variable target region set and the epigenetic target region set can have any of the features described for such sets in WO2020 / 160414, the entire contents of which are incorporated herein by reference. In some embodiments, the epigenetic target region set comprises a hypermethylated variable target region set. In some embodiments, the epigenetic target region set comprises a hypomethylated variable target region set. In some embodiments, the epigenetic target region set comprises a CTCF binding region. In some embodiments, the epigenetic target region set comprises a fragmented variable target region. In some embodiments, the epigenetic target region set comprises a transcription start site. In some embodiments, the set of epigenetic target regions comprises one or more of the following regions that may show local amplification in cancer: AR, BRAF, CCND1, CCND2, CCNE1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, KIT, KRAS, MET, MYC, PDGFRA, PIK3CA, and RAF1. For example, in some embodiments, the set of epigenetic target regions comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the above targets.

[0303] In some embodiments, the set of sequence variable target regions includes multiple regions known to undergo somatic mutations in cancer. In some aspects, the set of sequence variable target regions targets multiple different genes or genomic regions ("panels") selected so that a defined proportion of subjects with cancer exhibit genetic variants or tumor markers in one or more different genes or genomic regions within the panel. The panel can be selected so that the region for sequencing is limited to a fixed number of base pairs. The panel can be selected so that a desired amount of DNA is sequenced, for example, by adjusting the affinity and / or amount of probes described elsewhere herein. The panel can also be selected to achieve a desired sequence read depth. The panel can be selected to achieve a desired sequence read depth or sequence read coverage for a certain amount of sequenced base pairs. The panel can be selected to achieve a theoretical sensitivity, specificity, and / or accuracy for detecting one or more genetic variants in a sample.

[0304] The probe for detecting a group of regions can include those for detecting genomic regions of interest (hotspot regions).Information about chromatin structure can be considered when designing probe, and / or probe can be designed to maximize the possibility of capturing specific sites (for example, KRAS codon 12 and 13), and can be designed to optimize capture based on the analysis of cfDNA coverage and fragment size diversity, which are affected by nucleosome binding pattern and GC sequence composition.Region as used herein can also include non-hotspot regions that are optimized based on nucleosome position and GC model.

[0305] Examples of lists of genomic locations of interest can be found in Tables 3 and 4 of WO2020 / 160414. In some embodiments, the set of sequence variable target regions used in the methods of the disclosure includes at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or 70 of the genes in Table 3 of WO2020 / 160414. In some embodiments, the sequence variable target region set used in the method of the present disclosure comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or at least 73 parts of the genes in Table 4 of WO2020 / 160414.In addition, or alternatively, suitable target region sets can be obtained from literature.For example, Gale et al., PLoS One 13: e0194630 (2018), which is incorporated herein by reference, describes a group of 35 cancer-related gene targets that can be used as part or all of the sequence variable target region set. These 35 targets are AKT1, ALK, BRAF, CCND1, CDK2A, CTNNB1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, FGFR3, FOXL2, GATA3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MED12, MET, MYC, NFE2L2, NRAS, PDGFRA, PIK3CA, PPP2R1A, PTEN, RET, STK11, TP53, and U2AF1.

[0306] In some embodiments, the set of sequence variable target regions includes target regions from at least 10, 20, 30, or 35 cancer-associated genes, such as those listed above and in Tables 3 and 4 of WO2020 / 160414.

[0307] H. Sequencing Generally, sample proteins and / or nucleic acids, including adaptor-flanked nucleic acids, and / or nucleic acids generated (e.g., by an amplification step) from the oligonucleotide labels disclosed herein (e.g., the oligonucleotide labels illustrated in Figure 1A), can be subjected to sequencing with or without prior amplification. Sequencing methods include, for example, Edman degradation-based protein sequencing, mass spectrometry-based protein sequencing, Sanger sequencing, high-throughput sequencing, pyrosequencing, sequencing by synthesis, single molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing by ligation, sequencing by hybridization, Digital Gene Expression (Helicos), next-generation sequencing (NGS), Single Molecule Sequencing by Synthesis (SMSS) (Helicos), massively parallel sequencing, Clonal Single Molecule Array (Solexa), shotgun sequencing, Ion Torrent, Oxford Nanopore, Roche Genia, Maxam-Gilbert sequencing, primer walking, and sequencing using PacBio, SOLiD, Ion Torrent, or Nanopore platforms.

[0308] In some embodiments, sequencing includes detecting and / or distinguishing between unmodified and modified nucleobases. For example, single molecule real-time (SMRT) sequencing facilitates the direct detection of, for example, 5-methylcytosine and 5-hydroxymethylcytosine and unmodified cytosine. See, for example, Schatz., Nature Methods. 14(4): 347-348 (2017); and US 9,150,918. Sequencing reactions can be performed in various sample processing units, which may include multiple lanes, multiple channels, multiple wells, or other means for processing multiple sample sets substantially simultaneously. The sample processing unit may also include multiple sample chambers, allowing multiple runs to be processed simultaneously.

[0309] Sequencing reaction can be performed on one or more forms of nucleic acid, for example, those known to contain cancer or other disease markers.Sequencing reaction can also be performed on any nucleic acid fragment present in sample.In some embodiments, the sequencing coverage of genome can be less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, less than 95%, less than 99%, less than 99.9% or less than 100%.In some embodiments, sequencing reaction can produce at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70% or 80% sequencing coverage of genome. Sequencing coverage can be performed for at least 5, 10, 20, 70, 100, 200 or 500 different genes, or for up to 5000, 2500, 1000, 500 or 100 different genes.

[0310] Multiplex sequencing can be used to carry out simultaneous sequencing reactions.In some cases, cell-free nucleic acid can be sequenced with at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100,000 sequencing reactions.In other cases, cell-free nucleic acid can be sequenced with less than 1000, less than 2000, less than 3000, less than 4000, less than 5000, less than 6000, less than 7000, less than 8000, less than 9000, less than 10000, less than 50000, less than 100,000 sequencing reactions.Sequencing reactions can be carried out sequentially or simultaneously.Subsequent data analysis can be carried out on all or part of sequencing reactions. In some cases, data analysis can be performed on at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, or 100,000 sequencing reactions. In other cases, data analysis can be performed on less than 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, or 100,000 sequencing reactions. An exemplary read depth is 1000 to 50,000 reads per locus (base).

[0311] III. ADDITIONAL FEATURES OF CERTAIN DISCLOSED METHODS A. Sample The sample may be any biological sample isolated from a subject. The sample may be a bodily sample. The sample may include bodily tissues or fluids, such as known or suspected solid tumors, whole blood, platelets, serum, plasma, stool, red blood cells, white blood cells (leucocytes), endothelial cells, tissue biopsies, cerebrospinal fluid, synovial fluid, lymphatic fluid, ascites, interstitial or extracellular fluid, interstitial space fluid, gingival crevicular fluid, bone marrow, pleural fluid, pleural fluid, cerebrospinal fluid, saliva, mucus, sputum, semen, sweat, and urine. The sample is preferably a bodily fluid, particularly blood and its fractions, cerebrospinal fluid, pleural fluid, saliva, sputum, or urine. The sample may be in the form originally isolated from the subject or may have been subjected to further processing to remove or add components such as cells or to enrich one component for another. Therefore, preferred body fluids for analysis are plasma or serum, which may optionally contain cell-free nucleic acids.

[0312] In some embodiments, the sample contains one or more target proteins (e.g., first, second, third, fourth, and / or fifth target proteins, or higher ordinal target proteins), e.g., one or more target proteins containing one or more PTMs. In some embodiments, the population of nucleic acids is obtained from a serum, plasma, or blood sample from a subject suspected of having or previously diagnosed with neoplasia, tumor, precancerous condition, or cancer. The population contains nucleic acids with varying levels of sequence diversity, epigenetic diversity, and / or post-replicative or post-transcriptional modifications. Post-replicative modifications include, in particular, modifications of cytosine at the 5-position of the nucleobase, such as 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine.

[0313] A sample can be isolated or obtained from a subject and transported to a site for sample analysis. The sample can be stored and shipped at a desired temperature, for example, room temperature, 4°C, -20°C, and / or -80°C. The sample can be isolated or obtained from a subject at a site for sample analysis. The subject can be a human, mammal, animal, companion animal, service animal, or pet. The subject can have cancer, a precancerous condition, an infectious disease, transplant rejection, or other disease or disorder associated with an altered immune system. The subject can be free of cancer or detectable symptoms of cancer. The subject can be treated with one or more cancer therapies, such as any one or more of chemotherapy, antibodies, vaccines, or biologics. The subject can be in remission. The subject may or may not have been diagnosed with cancer or a predisposition to any cancer-related genetic mutation / disorder.

[0314] In some embodiments, the sample comprises plasma. The volume of plasma obtained may depend on the desired read depth of the region being sequenced. Exemplary volumes are 0.4 to 40 ml, 5 to 20 ml, and 10 to 20 ml. For example, the volume may be 0.5 ml, 1 ml, 5 ml, 10 ml, 20 ml, 30 ml, or 40 ml. The volume of plasma sampled may be 5 to 20 ml.

[0315] B. Capture part As discussed above, molecules such as proteins and / or nucleic acids in a sample can be subjected to a capture step, in which target proteins or molecules having target regions are captured and analyzed.Target capture can involve the use of a capture moiety, such as an oligonucleotide labeled with biotin, and a second moiety or binding partner, such as streptavidin, that binds to the capture moiety.In some embodiments, the capture moiety and binding partner can have higher and lower capture yields for different sets of target regions, for example, a set of sequence-variable target regions and a set of epigenetic target regions, as discussed elsewhere herein.Methods involving capture moieties are further described, for example, in U.S. Patent No. 9,850,523, issued on December 26, 2017, which is incorporated herein by reference.

[0316] Capture moieties include, but are not limited to, biotin, avidin, streptavidin, nucleic acids containing specific nucleotide sequences, haptens recognized by antibodies, and magnetically attractable particles. Extraction moieties can also be members of binding pairs, such as biotin / streptavidin or hapten / antibody. In some embodiments, the capture moiety bound to the analyte is captured by a binding partner attached to an isolatable moiety, such as a magnetically attractable particle or a large particle that can be sedimented by centrifugation. The capture moiety can be any type of molecule that allows affinity separation of nucleic acids bearing the capture moiety from nucleic acids lacking the capture moiety. Exemplary capture moieties are biotin, which allows affinity separation by binding to streptavidin bound or ligatable to a solid phase, or oligonucleotides, which allow affinity separation by binding to complementary oligonucleotides bound or ligatable to a solid phase.

[0317] C. Computer Systems The methods of the present disclosure can be implemented using or with the aid of a computer system. Figure 2 shows a computer system 201 programmed or otherwise configured to implement the methods of the present disclosure. The computer system 201 may coordinate various aspects of sample preparation, sequencing, and / or analysis. In some examples, the computer system 201 is configured to perform sample analysis, including sample preparation and nucleic acid sequencing (if applicable), e.g., according to any of the methods disclosed herein.

[0318] The computer system 201 includes a central processing unit (CPU, also referred to herein as a "processor" and a "computer processor") 205, which may be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system 201 also includes memory or memory locations 210 (e.g., random access memory, read-only memory, flash memory), electronic storage 215 (e.g., a hard disk), a communication interface 220 (e.g., a network adapter) for communicating with one or more other systems, and peripheral devices 225, such as cache, other memory, data storage, and / or an electronic display adapter. The memory 210, storage 215, interface 220, and peripheral devices 225 communicate with the CPU 205 via a bus (solid lines), such as a communications network or a motherboard. The storage 215 may be a data storage device (or data repository) for storing data. The computer system 201 may be operably coupled to a computer network 230 utilizing the communication interface 220. Computer network 230 may be the Internet, an Internet and / or extranet, or an intranet and / or extranet in communication with the Internet. Computer network 230, in some cases, is a telecommunications and / or data network. Computer network 230 may include one or more computer servers, which may enable distributed computing such as cloud computing. Computer network 230, in some cases, may leverage computer system 201 to implement a peer-to-peer network, which may enable devices coupled to computer system 201 to act as clients or servers.

[0319] CPU 205 may execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as memory 210. Examples of operations performed by CPU 205 may include fetch, decode, execute, and write-back.

[0320] The storage device 215 may store files, such as drivers, libraries, and saved programs. The storage device 215 may store programs and recorded sessions generated by users, as well as output(s) associated with the programs. The storage device 215 may store user data, such as user preferences and user programs. The computer system 201 may, in some cases, include one or more additional data storage devices located external to the computer system 201, for example, on a remote server with which the computer system 201 communicates over an intranet or the Internet. Data can be transferred from one location to another, for example, by using a communications network or physical data migration (e.g., using a hard drive, thumb drive, or other data storage mechanism).

[0321] Computer system 201 may communicate with one or more remote computer systems over network 230. In some embodiments, computer system 201 may communicate with a remote computer system of a user (e.g., an operator). Examples of remote computer systems include a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., an Apple® iPad®, a Samsung® Galaxy Tab), a telephone, a smartphone (e.g., an Apple® iPhone®, an Android®-enabled device, a Blackberry®), or a personal digital assistant. A user may access computer system 201 over network 230.

[0322] The methods described herein may be implemented by machine-executable (e.g., computer processor) code stored in an electronic storage location of the computer system 201, such as memory 210 or electronic storage 215. The machine-executable or machine-readable code may be provided in the form of software. During use, the code may be executed by the processor 205. In some cases, the code may be retrieved from storage 215 and stored in memory 210 for ready access by the processor 205.

[0323] In some circumstances, electronic storage device 215 may be omitted, with machine-executable instructions stored in memory 210.

[0324] In an aspect, the present disclosure provides a non-transitory computer-readable medium comprising computer-executable instructions that, when executed by at least one electronic processor, perform at least a portion of the methods described herein. For example, the method may include collecting a sample from a subject and, if necessary, fractionating the sample into sub-samples; pre-enriching post-translationally modified proteins, comprising contacting the sample or sub-sample with a first lectin that specifically binds to a first saccharide present in a post-translational modification (PTM) on one or more target proteins in the sample, thereby producing a first complex comprising the first lectin and the target protein; and separating the first complex from other components of the sample or sub-sample, thereby obtaining a first pre-enriched sub-sample; and determining the presence or level of at least one post-translationally modified target protein, comprising contacting the first pre-enriched sub-sample with a plurality of binding molecules, comprising a first binding molecule that specifically binds to a first epitope of the first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, wherein each of the first and second binding molecules comprises a label; and detecting the labels of the first and second binding molecules.

[0325] The code may be pre-compiled and configured for use on a machine having a processor adapted to execute the code, or it may be compiled at run time. The code may be supplied in a programming language that can be selected to enable the code to be executed in a pre-compiled or compiled-on-demand manner.

[0326] Aspects of the systems and methods provided herein, such as computer system 201, can be embodied in programming. Various aspects of the technology can be considered "products" or "articles of manufacture," generally in the form of machine (or processor) executable code and / or associated data embodied or embodied as some type of machine-readable medium. The machine-executable code can be stored in electronic storage, such as memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. A "storage" type medium can include any or all of the tangible memory of a computer, processor, etc., or its associated modules, e.g., various semiconductor memories, tape drives, disk drives, etc., that can provide non-transitory storage at any time for software programming.

[0327] All or portions of the software may sometimes be communicated over the Internet or various other telecommunications networks. Such communication may, for example, enable loading of the software from one computer or processor to another, e.g., from a management server or host computer to an application server computer platform. Thus, other types of media that may carry software elements include light waves, radio waves, and electromagnetic waves, such as those used across physical interfaces between local devices, through wired and optical networks, and by various air links. Physical elements that carry such waves, e.g., wired or wireless links, optical links, etc., may also be considered software-borne media. As used herein, except when limited to non-transitory tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0328] Thus, machine-readable media, such as computer-executable code, may take many forms, including but not limited to tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, any of the storage devices in any computer(s), such as those that can be used to implement, for example, the databases shown in the figures. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer-readable media include, for example, floppy disks, flexible disks, hard disks, magnetic tape, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punch cards, paper tape, any other physical storage media with a pattern of holes, RAM, ROM, PROMs and EPROMs, FLASH-EPROMs, any other memory chip or cartridge, a carrier wave carrying data or instructions, a cable or link which transports such a carrier wave, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer-readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0329] The computer system 201 may include or be in communication with an electronic display that includes a user interface (UI), for example, to provide one or more results of a sample analysis. Examples of UIs include, without limitation, graphical user interfaces (GUIs) and web-based user interfaces.

[0330] Additional details regarding computer systems and networks, databases, and computer program products can be found, for example, in Peterson, Computer Networks: A Systems Approach, Morgan Kaufmann, 5th Ed. (2011), Kurose, Computer Networking: A Top-Down Approach, Pearson, 7 th Ed. (2016), Elmasri, Fundamentals of Database Systems, Addison Wesley, 6th Ed. (2010), Coronel, Database Systems: Design, Implementation, & Management, Cengage Learning, 11 th Ed. (2014), Tucker, Programming Languages, McGraw-Hill Science / Engineering / Math, 2nd Ed. (2006), and Rhoton, Cloud Computing Architected: Solution Design Handbook, Recursive Press (2011).

[0331] D. Application 1. Cancer and other diseases The methods can be used to diagnose the presence of a condition in a subject, such as cancer or a precancerous condition, characterize the condition (e.g., determine the stage of the cancer or the heterogeneity of the cancer), monitor the subject's response to receiving treatment for the condition (e.g., response to a chemotherapeutic or immunotherapeutic agent), assess the subject's prognosis (e.g., predict survival outcomes in subjects with cancer), determine the subject's risk of developing a condition, predict the subsequent course of a condition in a subject, determine cancer metastasis or recurrence (or risk of cancer metastasis or recurrence) in a subject, and / or monitor the subject's health as part of a preventative health monitoring program (e.g., determine whether and / or when a subject requires further diagnostic screening). The present disclosure may also be useful in determining the effectiveness of particular treatment options. In a successful treatment option, if the treatment is successful, more cancer cells may die and DNA may be excreted, so that, for example, the amount of target proteins, the number and / or type of PTMs for one or more of the target proteins, copy number variation, rare mutations, and / or cancer-related epigenetic signatures (e.g., hypermethylated or hypomethylated regions) detected in a sample from the subject, for example, in the subject's blood (e.g., in DNA isolated from a buffy coat sample or any other sample containing cells, for example, in a blood sample from the subject (e.g., a whole blood sample, a leukapheresis sample, or a PBMC sample)), may increase; or, for example, a successful treatment may result in an increase or decrease in the quantity of a particular protein in the blood, while an unsuccessful treatment may result in no change. In other examples, this may not occur. In another example, a particular treatment option can be correlated with the cancer's profile (e.g., that of the target protein and / or genetic profile) over time. This correlation may be useful for selecting a treatment.

[0332] Additionally, if the cancer is observed to be in remission after treatment, the method can be used to monitor the possibility of residual disease or the possibility of disease recurrence.

[0333] In some embodiments, the method is used for screening cancer or in a method for screening cancer.For example, the sample can be from a subject who has not been diagnosed with cancer before.In some embodiments, the subject can have or not have cancer.In some embodiments, the subject can have or not have early stage cancer.In some embodiments, the subject has one or more risk factors for cancer, such as tobacco use (e.g., smoking), being overweight or obese, having a high body mass index (BMI), being elderly, having poor nutrition, having a high alcohol consumption, or having a family history of cancer.

[0334] In some embodiments, the subject has been using tobacco, for example, for at least 1 year, 5 years, 10 years, or 15 years. In some embodiments, the subject has a high BMI, for example, a BMI of 25 or higher, 26 or higher, 27 or higher, 28 or higher, 29 or higher, or 30 or higher. In some embodiments, the subject is at least 40, 45, 50, 55, 60, 65, 70, 75, or 80 years old. In some embodiments, the subject is nutritionally deficient, for example, has a high consumption of one or more of red meat and / or processed meat, trans fat, saturated fat, and refined sugar, and / or a low consumption of fruits and vegetables, complex carbohydrates, and / or unsaturated fat. High and low consumption can be defined as above or below, respectively, the recommendations in the Dietary Guidelines for Americans 2020-2025, available at, for example, www.dietaryguidelines.gov / sites / default / files / 2021-03 / Dietary_Guidelines_for_Americans-2020-2025.pdf. In some embodiments, the subject has a high alcohol consumption, e.g., an average of at least 3, 4, or 5 drinks per day (where 1 drink is about 1 ounce or 30 mL of 80-proof hard liquor or equivalent). In some embodiments, the subject has a family history of cancer, e.g., at least one, two, or three blood relatives have been previously diagnosed with cancer. In some embodiments, a blood relative is at least a third-degree relative (e.g., a great-grandparent, a great-aunt or great-uncle, or a cousin), at least a second-degree relative (e.g., a grandparent, an aunt or uncle, or a sibling sharing one parent), or a first-degree relative (e.g., a sibling sharing one or both parents).

[0335] In some embodiments, the methods and systems disclosed herein can be used to identify customized or targeted therapies for treating a given disease or condition in a patient based on the presence of one or more proteins of interest (i.e., one or more target proteins), the presence and / or absence of one or more PTMs to one or more target proteins, and / or the classification of nucleic acid variants as being of somatic or germline origin. Generally, the disease under consideration is a type of cancer. Non-limiting examples of such cancers include biliary tract cancer, bladder cancer, transitional cell carcinoma, urothelial carcinoma, brain cancer, glioma, astrocytoma, breast cancer, metaplastic carcinoma, cervical cancer, cervical squamous cell carcinoma, rectal cancer, colorectal cancer, colon cancer, hereditary nonpolyposis colorectal cancer, colorectal adenocarcinoma, gastrointestinal stromal tumor (GIST), endometrial cancer, endometrial stromal sarcoma, esophageal cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, intraocular melanoma, uveal melanoma, gallbladder cancer, gallbladder adenocarcinoma, renal cell carcinoma, renal clear cell carcinoma, transitional cell carcinoma, urothelial carcinoma, Wilms' tumor, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CHL), and leukemia-associated malignant tumors (LEMTs). CMML), liver cancer, liver carcinoma, hepatoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, B-cell lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, T-cell lymphoma, non-Hodgkin's lymphoma, precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma, multiple myeloma, nasopharyngeal carcinoma (NPC), neuroblastoma, oropharyngeal cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, pseudopapillary neoplasm, acinar cell carcinoma, prostate cancer, prostate adenocarcinoma, skin cancer, melanoma, malignant melanoma, cutaneous melanoma, small intestine cancer, gastric cancer, gastrointestinal stromal tumor (GIST), uterine cancer, or uterine sarcoma.The type and / or stage of cancer can be detected from genetic variations including mutations, rare mutations, indels, rearrangements, copy number variations, transversions, translocations, recombinations, inversions, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structural changes, gene fusions, chromosomal fusions, gene truncations, gene amplifications, gene duplications, chromosomal damage, DNA damage, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns, and abnormal changes in nucleic acid 5-methylcytosine.

[0336] Target protein and genetic data can also be used to characterize specific forms of cancer. Cancers are often heterogeneous in both composition and stage classification. Genetic profile data can enable the characterization of specific subtypes of cancer, which may be important in the diagnosis or treatment of that specific subtype. This information can also provide subjects or practitioners with clues regarding the prognosis of specific types of cancer, allowing either subjects or practitioners to adapt treatment options as the disease progresses. Some cancers can progress to become more aggressive and genetically unstable. Other cancers can remain benign, inactive, or dormant. The systems and methods of the present disclosure can be useful in determining disease progression.

[0337] Furthermore, the methods of the present disclosure can be used to characterize the heterogeneity of an abnormal condition in a subject. Such a method can include, for example, generating a genetic profile of extracellular molecules and polynucleotides from the subject, where the genetic profile includes multiple data obtained from analysis of copy number variation and rare mutations. In some embodiments, the abnormal condition is cancer. In some embodiments, the abnormal condition can result in a heterogeneous genomic population. In the example of cancer, it has been found that some tumors contain tumor cells at different stages of cancer. In other examples, the heterogeneity can include multiple foci of disease. Again, in the example of cancer, there can be multiple tumor foci, perhaps one or more of which are the result of metastasis that has spread from the primary site. The tissue(s) of origin can be useful for identifying organs affected by cancer, including the primary cancer and / or metastatic tumors.

[0338] The method can be used to generate a profile, fingerprint, or data set that is a summary of target protein and gene information from different cells in a heterogeneous disease. This data set can include, alone or in combination, protein levels (e.g., of one or more target proteins), amount and / or type of PTM to one or more proteins, copy number variation, epigenetic diversity, and mutation analysis.

[0339] This method can be used to diagnose, prognose, monitor or observe cancer, precancerous condition or other disease.In some embodiments, the method herein does not involve diagnosing, prognosing or monitoring fetus, and therefore is not intended for non-invasive prenatal testing.In other embodiments, these methodologies can be used in pregnant subjects to diagnose, prognose, monitor or observe cancer or other disease in unborn subjects, where DNA and other polynucleotides may be co-circulating with maternal molecules.

[0340] Non-limiting examples of other genetically based diseases, disorders, or conditions that may be assessed, if desired, using the methods and systems disclosed herein include achondroplasia, alpha 1 antitrypsin deficiency, antiphospholipid syndrome, autism, autosomal dominant polycystic kidney disease, Charcot-Marie-Tooth (CMT), cri-a-cat, Crohn's disease, cystic fibrosis, Dercum's disease, Down's syndrome, Duane's syndrome, Duchenne muscular dystrophy, factor V Leiden thrombophilia, familial hypercholesterolemia, familial Mediterranean fever, and fragile X syndrome. These include: Gaucher's disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington's disease, Klinefelter's syndrome, Marfan's syndrome, myotonic dystrophy, neurofibromatosis, Noonan's syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland anomaly, porphyria, progeria, retinitis pigmentosa, severe combined immunodeficiency (SCID), sickle cell disease, spinal muscular atrophy, Tay-Sachs disease, thalassemia, trimethylaminuria, Turner's syndrome, palatocardiofacial syndrome, WAGR syndrome, and Wilson's disease.

[0341] In some embodiments, the methods described herein include detecting the presence or absence of one or more target proteins and / or one or more PTMs for one or more target proteins originating from or derived from tumor cells at a preselected time point after a previous cancer treatment in a subject previously diagnosed with cancer. DNA originating from or derived from the tumor cells can also be detected. The method may further include determining a cancer recurrence score indicating the presence or absence of target proteins and, if applicable, DNA originating from or derived from the subject's tumor cells.

[0342] When determining the cancer recurrence score, the score can be further used to determine the cancer recurrence status.The cancer recurrence status can be, for example, when the cancer recurrence score is above a predetermined threshold, that there is a risk of cancer recurrence.The cancer recurrence status can be, for example, when the cancer recurrence score is above a predetermined threshold, that there is a low or lower risk of cancer recurrence.In certain embodiments, a cancer recurrence score equal to a predetermined threshold can result in a cancer recurrence status of either there is a risk of cancer recurrence or a low or lower risk of cancer recurrence.

[0343] In some embodiments, the cancer recurrence score is compared to a predetermined cancer recurrence threshold, and the subject is classified as a candidate for subsequent cancer treatment if the cancer recurrence score is above the cancer recurrence threshold, or as not a candidate for treatment if the cancer recurrence score is below the cancer recurrence threshold. In certain embodiments, a cancer recurrence score equal to the cancer recurrence threshold may result in classification as either a candidate for subsequent cancer treatment or as not a candidate for treatment.

[0344] The methods discussed above may further include any suitable feature(s) described elsewhere herein, including the sections relating to methods for determining the risk of cancer recurrence in a subject and / or classifying a subject as a candidate for subsequent cancer treatment.

[0345] 2. Methods for determining the risk of cancer recurrence in a subject and / or classifying a subject as a candidate for subsequent cancer treatment In some embodiments, the methods provided herein are methods for determining the risk of cancer recurrence in a subject. In some embodiments, the methods provided herein are methods for classifying a subject as a candidate for a subsequent cancer treatment.

[0346] Any of these methods may include collecting a sample from a subject diagnosed with cancer at one or more preselected time points after one or more previous cancer treatments for the subject. The subject may be any of the subjects described herein. The sample may include proteins from dead or dying cells. The sample may include DNA, for example, cfDNA. DNA may be obtained from a tissue sample.

[0347] Any of these methods may include contacting a sample or a subsample thereof with at least one binding molecule and detecting the presence or level of one or more target proteins and / or one or more PTMs for one or more target proteins according to any of the embodiments described herein. The method may further include capturing a plurality of sets of target regions from DNA from the subject, the plurality of sets of target regions including a set of sequence-variable target regions and / or a set of epigenetic target regions, thereby generating a captured set of DNA molecules. The capturing step may be performed according to any of the embodiments described elsewhere herein. Any of these methods may include sequencing the captured DNA molecules, thereby generating a set of sequence information. The captured DNA molecules of the set of sequence-variable target regions may be sequenced to a deeper sequencing depth than the captured DNA molecules of the set of epigenetic target regions. Any of these methods may include, at a preselected time point, detecting the presence or absence of DNA originating from or derived from tumor cells using the set of sequence information. Detection of the presence or absence of DNA originating or derived from tumor cells can be carried out according to any of the embodiments thereof described elsewhere herein.

[0348] In any of such methods, the previous cancer treatment may include surgery, administration of a therapeutic composition, and / or chemotherapy.

[0349] The method for determining the risk of cancer recurrence in a subject may include determining a cancer recurrence score for the subject, the cancer recurrence score indicating the presence, absence, or amount of one or more target proteins and / or one or more nucleic acids originating from or derived from tumor cells, and / or the presence or amount of one or more PTMs for one or more target proteins originating from or derived from tumor cells. The cancer recurrence score can further be used to determine a cancer recurrence status. For example, if the cancer recurrence score is above a predetermined threshold, the cancer recurrence status may be, for example, a risk of cancer recurrence. For example, if the cancer recurrence score is above a predetermined threshold, the cancer recurrence status may be, for example, a low or lower risk of cancer recurrence. In certain embodiments, a cancer recurrence score equal to a predetermined threshold may result in a cancer recurrence status of either a risk of cancer recurrence or a low or lower risk of cancer recurrence.

[0350] A method for classifying a subject as a candidate for subsequent cancer treatment may include comparing the subject's cancer recurrence score to a predetermined cancer recurrence threshold, thereby classifying the subject as a candidate for subsequent cancer treatment if the cancer recurrence score is above the cancer recurrence threshold, or as not a candidate for treatment if the cancer recurrence score is below the cancer recurrence threshold. In certain embodiments, a cancer recurrence score equal to the cancer recurrence threshold may result in classification as a candidate for subsequent cancer treatment or as not a candidate for treatment. In some embodiments, the subsequent cancer treatment includes administration of chemotherapy or a therapeutic composition.

[0351] Any of these methods can include determining the disease-free survival (DFS) period for the subject based on cancer recurrence score.For example, the DFS period can be 1 year, 2 years, 3 years, 4 years, 5 years or 10 years.

[0352] In some embodiments, the set of sequence information comprises a sequence variable target region sequence, and determining the cancer recurrence score may comprise determining at least a first subscore indicative of the level of a particular immune cell type, SNV, insertion / deletion, CNV and / or fusion present within the sequence variable target region sequence.

[0353] In some embodiments, a number of mutations in the sequence variable target region selected from 1, 2, 3, 4, or 5 is sufficient to result in a cancer recurrence score in which the first subscore is classified as positive for cancer recurrence. In some embodiments, the number of mutations is selected from 1, 2, or 3.

[0354] In any embodiment in which the Cancer Recurrence Score is classified as positive for cancer recurrence, the subject's cancer recurrence status may be at risk for cancer recurrence and / or the subject may be classified as a candidate for subsequent cancer treatment.

[0355] In some embodiments, the cancer is any one of the types of cancer described elsewhere herein, for example, colorectal cancer.

[0356] 3. Methods for monitoring cancer in a subject over time; sample collection at two or more time points In some embodiments, the methods can be used to monitor one or more aspects of a condition in a subject over time, such as the subject's response to receiving treatment for the condition (e.g., response to a chemotherapeutic or immunotherapeutic agent), the severity of the condition in a subject (e.g., the stage of cancer), the recurrence of the condition (e.g., cancer), and / or the subject's risk of developing a condition (e.g., cancer), and / or to monitor the health of a subject as part of a preventative health monitoring program (e.g., to determine whether and / or when the subject requires further diagnostic screening). In some embodiments, monitoring involves analysis of at least two samples collected from the subject at at least two different time points as described herein.

[0357] The method according to the present disclosure can also be useful for predicting a subject's response to a particular treatment option. A successful treatment option may result in an increase or decrease in the quantity of one or more target proteins and / or the quantity and / or type of one or more PTMs for one or more target proteins (e.g., in the blood), while an unsuccessful treatment may result in no change. In other examples, this may not occur. In another example, a particular treatment option can be correlated with the cancer profile (e.g., target protein and / or genetic profile) over time. This correlation can be useful for selecting a treatment for a subject.

[0358] The disclosed methods may include assessing (e.g., quantifying) and / or interpreting protein(s) present in one or more samples comprising cells or blood samples (e.g., buffy coat samples, whole blood samples, leukoreduction samples, or PBMC samples) collected from a subject at one or more time points relative to a selected baseline value or reference standard (or a selected set of baseline values ​​or reference standards). The baseline value or reference standard may be the quantity of one or more target proteins and / or the quantity or type of one or more PTMs for one or more target proteins (e.g., the average quantity or range of quantities of protein(s) present in at least two samples) measured in one or more samples collected from the subject at one or more time points, e.g., before undergoing treatment, before diagnosis of a condition (e.g., cancer), or as part of a preventative health monitoring program. A baseline value or reference standard may be the quantity of a protein(s) (e.g., the average quantity or range of quantities of a protein(s) present in at least two samples) measured in one or more samples collected at one or more time points from one or more subjects without a condition (e.g., healthy subjects without cancer), one or more subjects who have responded favorably to a treatment, or one or more subjects not receiving treatment. In certain embodiments, the baseline value or reference standard utilized is a standard or profile derived from a single reference subject. In other embodiments, the baseline value or reference standard utilized is a standard or profile derived from average data from multiple reference subjects. In various embodiments, the reference standard may be a single value, an average, a mean, a numerical mean or range of numerical means, a numerical pattern, or a graphical pattern generated from quantitative data of cell types from a single reference subject or multiple reference subjects. The selection of a particular baseline value or reference standard, or the selection of one or more reference subjects, depends, for example, on the use of the methods described herein made by a research scientist or clinician (e.g., physician).

[0359] In some embodiments, one or more samples (e.g., a sample comprising cells, or a blood sample (e.g., a buffy coat sample, a whole blood sample, a leukoreduction sample, or a PBMC sample) can be collected from a subject at two or more time points to assess changes in a protein(s) (e.g., changes in the quantity of a protein(s) or changes in one or more modifications (e.g., one or more post-translational modifications) of a protein(s)) between two or more time points. In some embodiments, the sample collected at a first time point is a tissue sample or a blood sample, and the sample collected at a subsequent time point (e.g., a second time point) is a blood sample. In some embodiments, the sample collected at a first time point is a tissue sample, and the sample collected at a subsequent time point (e.g., a second time point) is a blood sample. By monitoring protein(s) in samples collected from a subject at two or more time points and identifying differences between the protein(s), the method can also be used to diagnose, for example, the presence of a condition (e.g., cancer). The presence or absence of a condition, the subject's response to a treatment, one or more characteristics of a condition (e.g., the stage of cancer) in the subject, the recurrence of a condition (e.g., cancer), and / or the subject's risk of developing a condition (e.g., cancer) can be determined. Thus, in some embodiments, methods are provided that compare the quantity of a protein(s) present in at least one sample (e.g., at least one whole blood sample, buffy coat sample, leukopheresis sample, or PBMC sample) collected from a subject at one or more time points (e.g., before receiving a treatment) with the quantity of a protein(s) present in at least one sample collected from the subject at one or more different time points (e.g., after receiving a treatment). The disclosed methods can enable patient-specific monitoring, such that, for example, differences in protein quantity and / or protein modification between samples collected from a subject at different time points can indicate changes (e.g., the presence or absence of a condition, response to a treatment, prognosis, etc.) that are meaningful with respect to the subject but may still fall within the normal range for the general healthy population.

[0360] As disclosed herein, methods are provided for monitoring one or more aspects of a condition in a subject over time, including, but not limited to, the subject's response to receiving a treatment for the condition (e.g., response to a chemotherapeutic or immunotherapeutic agent). In certain embodiments, one or more samples are collected from the subject at least 1-10, at least 1-5, at least 2-5, or at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 time points before the subject receives the treatment. In certain embodiments, one or more samples are collected from the subject at least 1-10, at least 1-5, at least 2-5, or at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 time points after the subject receives the treatment. Collection of samples from the subject can continue during and / or after the treatment to monitor the subject's response to the treatment.

[0361] In some embodiments, samples are not collected from a subject prior to diagnosis of a condition (e.g., cancer) or prior to receiving treatment. In such embodiments, the subject's response to treatment, or the course or stage of a condition (e.g., cancer) in a subject, is monitored over time, and cell types are compared among samples obtained at least 2-10, at least 2-5, at least 3-6, or at least 2, e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 time points collected after the subject is diagnosed and / or after the subject receives treatment. Collection of samples from the subject can continue during and / or after treatment to monitor the subject's response to treatment.

[0362] In some embodiments of the disclosed methods, one or more samples, e.g., cell-containing or blood samples (e.g., one or more whole blood, buffy coat, leukopheresis, or PBMC samples), are collected from a subject at least once per year, e.g., about 1-12 times or about 2-6 times per year, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times per year. In other embodiments, one or more samples are collected from a subject less than once per year, e.g., about once every 13 months, about once every 14 months, about once every 15 months, about once every 16 months, about once every 17 months, about once every 18 months, about once every 19 months, about once every 20 months, about once every 21 months, about once every 22 months, about once every 23 months, or about once every 24 months. In some embodiments, one or more samples are collected from a subject about once every 1 to 5 years or about once every 1 to 2 years, e.g., about every year, about every 1.5 years, about every 2 years, about every 2.5 years, about every 3 years, about every 3.5 years, about every 4 years, about every 4.5 years, or about every 5 years.

[0363] In other embodiments of the disclosed methods, one or more samples, e.g., one or more samples comprising cells, one or more blood samples, e.g., one or more buffy coat samples, whole blood samples, leukoreduction samples, or PBMC samples, are collected from a subject at least once per week, e.g., on 1-4 days, 1-2 days, or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days per week. In certain embodiments, one or more samples are collected from a subject at least once per month, e.g., 1-15 times, 1-10 times, 2-5 times, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, 11 times, 12, 13, 14, or 15 times per month. In other embodiments, one or more samples are collected from a subject every month, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, or every twelve months. In some embodiments, one or more samples are collected from a subject at least once per day, for example, once, twice, three times, four times, five times, or six times per day. The selection of one or more sample collection time points (e.g., frequency of sample collection), or the selection of the number of samples collected at each time point, depends on the use to which the methods described herein are made, for example, by a research scientist or a clinician (e.g., a physician).

[0364] 4. Treatment and Related Administration In certain embodiments, the methods disclosed herein relate to identifying and administering a therapy, e.g., a customized therapy, to a patient or subject. In some embodiments, determining the level of a particular target protein and / or the level and / or type of one or more PTMs to the target protein facilitates the selection of an appropriate treatment.

[0365] In some embodiments, the patient or subject has a given disease, disorder or condition. Essentially any cancer treatment (for example, surgery, radiation therapy, chemotherapy, immunotherapy, etc.) can be included as part of these methods. In certain embodiments, the treatment administered to the subject comprises at least one chemotherapeutic agent. In some embodiments, chemotherapeutic agents may include alkylating agents (e.g., but not limited to, chlorambucil, cyclophosphamide, cisplatin, and carboplatin), nitrosoureas (e.g., but not limited to, carmustine and lomustine), antimetabolites (e.g., but not limited to, Fluorauracil, methotrexate, and fludarabine), plant alkaloids and natural products (e.g., but not limited to, vincristine, paclitaxel, and topotecan), antitumor antibiotics (e.g., but not limited to, bleomycin, doxorubicin, and mitoxantrone), hormonal agents (e.g., but not limited to, prednisone, dexamethasone, tamoxifen, and leuprolide), and biological response modifiers (e.g., but not limited to, Herceptin and Avastin, Erbitux, and Rituxan). In some embodiments, the chemotherapy administered to the subject may include FOLFOX or FOLFIRI. In certain embodiments, treatment comprising at least one PARP inhibitor can be administered to the subject.In certain embodiments, PARP inhibitors can include, among others, OLAPARIB, TALAZOPARIB, RUCAPARIB, NIRAPARIB (trade name ZEJULA).Generally, treatment comprises at least one immunotherapy (or immunotherapeutic agent).Immunotherapy generally refers to the method of enhancing immune response to a given cancer type.In certain embodiments, immunotherapy refers to the method of enhancing T cell response to tumor or cancer.

[0366] In some embodiments, the treatment is customized based on the somatic or germline origin of the nucleic acid variant. In some embodiments, essentially any cancer treatment (for example, surgical therapy, radiation therapy, chemotherapy, and / or the like) can be included as part of these methods. Generally, the customized treatment includes at least one immunotherapy (or immunotherapeutic agent). Immunotherapy generally refers to a method of enhancing the immune response to a given cancer type. In certain embodiments, immunotherapy refers to a method of enhancing the T cell response to tumor or cancer.

[0367] In some embodiments, immunotherapy or immunotherapeutic agents target immune checkpoint molecules. Certain tumors can evade the immune system by exploiting immune checkpoint pathways. Therefore, targeting immune checkpoints has emerged as an effective approach to block tumors' ability to evade the immune system and activate anti-tumor immunity against certain cancers. Pardoll, Nature Reviews Cancer, 2012, 12: 252-264.

[0368] In certain embodiments, the immune checkpoint molecule is an inhibitory molecule that reduces signals involved in T cell responses to antigens. For example, CTLA4 is expressed on T cells and plays a role in downregulating T cell activation by binding to CD80 (also known as B7.1) or CD86 (also known as B7.2) on antigen-presenting cells. PD-1 is another inhibitory checkpoint molecule expressed on T cells. PD-1 limits T cell activity in peripheral tissues during inflammatory responses. Furthermore, PD-1 ligands (PD-L1 or PD-L2) are commonly upregulated on the surface of many different tumors, resulting in downregulation of anti-tumor immune responses in the tumor microenvironment. In certain embodiments, the inhibitory immune checkpoint molecule is CTLA4 or PD-1. In other embodiments, the inhibitory immune checkpoint molecule is a PD-1 ligand, such as PD-L1 or PD-L2. In other embodiments, the inhibitory immune checkpoint molecule is a CTLA4 ligand, such as CD80 or CD86. In other embodiments, the inhibitory immune checkpoint molecule is lymphocyte activation gene 3 (LAG3), killer cell immunoglobulin-like receptor (KIR), T-cell membrane protein 3 (TIM3), galectin 9 (GAL9), or adenosine A2a receptor (A2aR).

[0369] Antagonists that target these immune checkpoint molecules can be used to enhance antigen-specific T cell responses against certain cancers. Thus, in certain embodiments, the immunotherapy or immunotherapeutic agent is an antagonist of an inhibitory immune checkpoint molecule. In certain embodiments, the inhibitory immune checkpoint molecule is PD-1. In certain embodiments, the inhibitory immune checkpoint molecule is PD-L1. In certain embodiments, the antagonist of an inhibitory immune checkpoint molecule is an antibody (e.g., a monoclonal antibody). In certain embodiments, the antibody or monoclonal antibody is an anti-CTLA4, anti-PD-1, anti-PD-L1, or anti-PD-L2 antibody. In certain embodiments, the antibody is a monoclonal anti-PD-1 antibody. In some embodiments, the antibody is a monoclonal anti-PD-L1 antibody. In certain embodiments, the monoclonal antibody is a combination of an anti-CTLA4 antibody and an anti-PD-1 antibody, an anti-CTLA4 antibody and an anti-PD-L1 antibody, or an anti-PD-L1 antibody and an anti-PD-1 antibody. In certain embodiments, the anti-PD-1 antibody is one or more of pembrolizumab (Keytruda®) or nivolumab (Opdivo®). In certain embodiments, the anti-CTLA4 antibody is ipilimumab (Yervoy®). In certain embodiments, the anti-PD-L1 antibody is one or more of atezolizumab (Tecentriq®), avelumab (Bavencio®), or durvalumab (Imfinzi®).

[0370] In certain embodiments, the immunotherapy or immunotherapeutic agent is an antagonist (e.g., an antibody) against CD80, CD86, LAG3, KIR, TIM3, GAL9, or A2aR. In other embodiments, the antagonist is a soluble version of an inhibitory immune checkpoint molecule, e.g., a soluble fusion protein comprising the extracellular domain of an inhibitory immune checkpoint molecule and the Fc domain of an antibody. In certain embodiments, the soluble fusion protein comprises the extracellular domain of CTLA4, PD-1, PD-L1, or PD-L2. In some embodiments, the soluble fusion protein comprises the extracellular domain of CD80, CD86, LAG3, KIR, TIM3, GAL9, or A2aR. In one embodiment, the soluble fusion protein comprises the extracellular domain of PD-L2 or LAG3.

[0371] In certain embodiments, the immune checkpoint molecule is a costimulatory molecule that amplifies signals involved in T cell responses to antigens. For example, CD28 is a costimulatory receptor expressed on T cells. When a T cell binds to an antigen through its T cell receptor, CD28 binds to CD80 (also known as B7.1) or CD86 (also known as B7.2) on an antigen-presenting cell, amplifying T cell receptor signaling and promoting T cell activation. Because CD28 binds to the same ligands (CD80 and CD86) as CTLA4, CTLA4 can counteract or regulate the costimulatory signaling mediated by CD28. In certain embodiments, the immune checkpoint molecule is a costimulatory molecule selected from CD28, inducible T cell costimulator (ICOS), CD137, OX40, or CD27. In other embodiments, the immune checkpoint molecule is a ligand for a costimulatory molecule, including, for example, CD80, CD86, B7RP1, B7-H3, B7-H4, CD137L, OX40L, or CD70.

[0372] Agonists targeting these costimulatory checkpoint molecules can be used to enhance antigen-specific T cell responses to certain cancers. Thus, in certain embodiments, the immunotherapy or immunotherapeutic agent is an agonist of a costimulatory checkpoint molecule. In certain embodiments, the agonist of a costimulatory checkpoint molecule is an agonist antibody, preferably a monoclonal antibody. In certain embodiments, the agonist antibody or monoclonal antibody is an anti-CD28 antibody. In other embodiments, the agonist antibody or monoclonal antibody is an anti-ICOS, anti-CD137, anti-OX40, or anti-CD27 antibody. In other embodiments, the agonist antibody or monoclonal antibody is an anti-CD80 antibody, anti-CD86 antibody, anti-B7RP1 antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-CD137L antibody, anti-OX40L antibody, or anti-CD70 antibody.

[0373] In certain embodiments, the somatic or germline origin status of nucleic acid variants from sample from subject can be compared with the database of comparable results from reference population to identify customized or targeted treatment for this subject.Generally, reference population comprises patients with the same cancer or disease type as subject and / or patients who are undergoing or have undergone the same treatment as subject.If nucleic acid variants and comparable results meet certain classification criteria (for example, are substantially or approximately identical), customized or targeted treatment(s) can be identified.

[0374] In certain embodiments, the customized therapy described herein is generally administered parenterally (e.g., intravenously or subcutaneously). Pharmaceutical compositions containing immunotherapeutic agents are generally administered intravenously. Certain therapeutic agents are administered orally. However, the customized therapy (e.g., immunotherapeutic agent, etc.) may also be administered by methods such as buccal, sublingual, rectal, vaginal, intraurethral, ​​topical, intraocular, intranasal, and / or intraauricular administration, and administration may include tablets, capsules, granules, aqueous suspensions, gels, sprays, suppositories, salves, ointments, etc.

[0375] IV. Kit Kits comprising the compositions described herein are also provided. The kits may be for use in carrying out the methods described herein. In some embodiments, the kits comprise one or more target protein-binding and / or PTM-binding molecules. In some embodiments, the marker-binding molecule comprises a label or capture moiety. In some embodiments, the kits comprise a solid support linked to a binding partner of the capture moiety. In some embodiments, the kits comprise one or more target protein-binding molecules. In some embodiments, the kits comprise reagents for detecting the presence or level of a target protein.

[0376] In some embodiments, the kit further comprises an agent that recognizes methylcytosine in DNA. In some such embodiments, the agent is an antibody or a methyl-binding protein or methyl-binding domain. In some embodiments, the kit comprises a target-specific probe that specifically binds to a set of epigenetic and / or sequence-variable target regions. In some such embodiments, the target-specific probe comprises a capture moiety. In some embodiments, the kit comprises a solid support linked to a binding partner of the capture moiety. In some embodiments, the kit comprises an adaptor. In some embodiments, the kit comprises PCR primers, where the PCR primers anneal to the target region or the adaptor. In some embodiments, the kit comprises additional elements described elsewhere herein. In some embodiments, the kit comprises instructions for performing the methods described herein.

[0377] The kit includes ALK, APC, BRAF, CDKN2A, EGFR, ERBB2, FBXW7, KRAS, MYC, NOTCH1, NRAS, PIK3CA, PTEN, RBI, TP53, MET, AR, ABLl, AKTl, ATM, CDHl, CSFIR, CTNNBl, ERBB4, EZH2, FGFRl, FGFR2, FGFR3, FLT3, GNA11, GNAQ, G The gene expression vector may further comprise a plurality of oligonucleotide probes that selectively hybridize to at least 5, 6, 7, 8, 9, 10, 20, 30, 40 or all of the genes selected from the group consisting of NAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, MLH1, MPL, NPM1, PDGFRA, PROC, PTPN11, RET, SMAD4, SMARCB1, SMO, SRC, STK11, VHL, TERT, CCND1, CDK4, CDKN2B, RAF1, BRCA1, CCND2, CDK6, NF1, TP53, ARID 1 A, BRCA2, CCNE1, ESR1, RIT1, GATA3, MAP2K1, RHEB, ROS1, ARAF, MAP2K2, NFE2L2, RHOA, and NTRK1. The number of genes to which the oligonucleotide probe can selectively hybridize can vary. For example, the number of genes can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54. The kit may include a container containing a plurality of oligonucleotide probes and instructions for carrying out any of the methods described herein.

[0378] The kit may include at least four, five, six, seven, or eight different library adapters with distinct molecular barcodes and identical sample barcodes. The library adapters may not be sequencing adapters. For example, the library adapters do not include a flow cell sequence for sequencing or a sequence that enables hairpin loop formation. Different variations and combinations of molecular barcodes and sample barcodes are described throughout and are applicable to the kit. Furthermore, in some cases, the adapters are not sequencing adapters. Furthermore, the adapters provided in the kit may also include sequencing adapters. The sequencing adapters may include a sequence that hybridizes to one or more sequencing primers. The sequencing adapters may further include a sequence that hybridizes to a solid support, such as a flow cell sequence. For example, the sequencing adapters may be flow cell adapters. The sequencing adapters may be attached to one or both ends of the polynucleotide fragments. In some cases, the kit may include at least eight different library adapters with distinct molecular barcodes and identical sample barcodes. The library adapter may not be a sequencing adapter. The kit may further include a sequencing adapter having a first sequence that selectively hybridizes to the adapter and a second sequence that selectively hybridizes to the flow cell sequence. In another example, the sequencing adapter may be hairpin-shaped. For example, the hairpin-shaped adapter may include a complementary double-stranded portion and a loop portion, where the double-stranded portion may be attached (e.g., ligated) to the double-stranded polynucleotide. Hairpin-shaped sequencing adapters may be attached to both ends of a polynucleotide fragment to generate a circular molecule that can be sequenced multiple times. The sequencing adapter may include one or more barcodes. For example, the sequencing adapter may include a sample barcode. The sample barcode may include a predetermined sequence. The sample barcode can be used to identify the source of the polynucleotide.The sample barcode can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleic acid bases (or any length described throughout), e.g., at least 8 bases. The barcode can be a continuous or non-contiguous sequence, as described above.

[0379] Library adapters can be blunt-ended and Y-shaped and can be less than or equal to 40 nucleobases in length, other variations of which can be found throughout and are applicable to the kits.

[0380] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided herein. While the present invention has been described with reference to the above specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, modifications, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it is to be understood that all aspects of the invention are not limited to the specific depictions, configurations, and relative proportions set forth herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the disclosed embodiments described herein can be employed in practicing the invention. Accordingly, it is intended that the present disclosure also cover such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0381] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art upon reading the present disclosure that various changes in form and details may be made therein without departing from the true scope of the disclosure and may be practiced within the purview of the appended claims. For example, all methods, systems, computer-readable media, and / or component features, steps, elements, or other aspects thereof may be used in various combinations.

[0382] All patents, patent applications, websites, other publications or documents, accession numbers, etc. cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be incorporated by reference. Where different versions of a sequence are associated with accession numbers at different times, the version associated with the accession number as of the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or, if applicable, the filing date of the priority application to which the accession number is referenced. Similarly, unless otherwise specified, where different versions of a publication, website, etc. are published at different times, the version published closest to the effective filing date of this application is meant. [Example]

[0383] Example 1 Analysis of circulating proteins using pre-enrichment This example analyzes patient samples using a blood-based assay to detect the presence or absence of cancer, using the workflow illustrated in Figure 1A. Proteins extracted from the sample are subjected to affinity chromatography using lectins that specifically bind to sialyl Lewis A and Tn antigens. Chromatography can be performed sequentially, where the flow-through from one pre-enrichment is used as input for the next pre-enrichment, or in parallel, where the proteins are divided into subsamples and pre-enriched separately, resulting in multiple pre-enriched separate fractions. Immunoassays are performed with the separate pre-enriched fractions using binding molecules, e.g., antibodies, specific for target proteins such as TP53, RB1, CTLA4, or PDL1, conjugated to an oligonucleotide label. For each target protein in each pre-enriched fraction, a pair of first and second binding molecules is used, binding to different epitopes and labeled with first and second oligonucleotides, respectively. The first and second oligonucleotides comprise two portions: 1) a first portion having a sequence unique to the type of binding molecule to which it is conjugated (e.g., a molecular barcode unique to an antibody specific for a particular target protein, such as an antibody specific for TP53, RB1, CTLA4, or PDL1), which also identifies the pre-enriched fraction in which the binding molecule is used, and 2) a second portion 3' to the first portion having a hybridization sequence. For each pair, the hybridization sequence of the first oligonucleotide is complementary to the hybridization sequence of the second oligonucleotide.

[0384] When the first and second oligonucleotides are within sufficiently close proximity to each other, they will hybridize, and the hybridized (double-stranded) oligonucleotide sequence is extended from the 3' end of the hybridization sequence using DNA polymerase.The extended oligonucleotides from multiple pre-enriched fractions are pooled, amplified, and sequenced using an Illumina sequencer or quantified by suitable procedures such as qPCR.For example, see Gong et al., Bioconjugate Chem. 2016, 27, 1, 217-225 for exemplary hybridization, extension, and sequencing-based detection procedures.

[0385] The sequence reads generated by sequencer are then analyzed using bioinformatics tools / algorithms.The molecular barcodes present in sequenced molecules are used to identify binding molecules and their epitopes, while also deconvoluting the target proteins or PTMs that they are close to.Compared to samples from healthy subjects, the quantification of sequence reads corresponding to post-translationally modified proteins in samples from patients, for example, the up-regulated post-translationally modified proteins in tumor cells, facilitates determining the likelihood that the patient has cancer. Example 2 Analysis of circulating proteins using simultaneous detection of target proteins and post-translational modifications

[0386] This example analyzes patient samples to detect the presence or absence of cancer using a blood-based assay using the workflow illustrated in Figure 1B. Proteins extracted from the sample are contacted with a first binding molecule, which is a lectin that specifically binds to sialyl Lewis A and is conjugated to a first oligonucleotide; a second binding molecule, which is an antibody that specifically binds to a target protein (e.g., TP53, RB1, CTLA4, or PDL1) and is conjugated to a second oligonucleotide; a third binding molecule (e.g., an antibody or lectin) that specifically binds to the Tn antigen and is conjugated to a third oligonucleotide; and an additional (fourth) binding molecule that specifically binds to an epitope of the target protein different from the epitope bound by the second molecule and is conjugated to a fourth oligonucleotide. Each of the first through fourth oligonucleotides contains two portions: 1) a first portion having a sequence (molecular barcode) unique to the binding molecule to which it is conjugated, and 2) a second portion 3' to the first portion that has a hybridization sequence. The hybridization sequence of each of the first, third and fourth oligonucleotides is the same and is complementary to the hybridization sequence of each of the second oligonucleotides.When the first and second oligonucleotides are within sufficiently close proximity to each other, they will hybridize, and the hybridized (double-stranded) oligonucleotide sequence is extended from the 3' end of the hybridization sequence using DNA polymerase.The same applies to the second and third oligonucleotides and the second and fourth oligonucleotides.The extended oligonucleotides are pooled, amplified, and sequenced using an Illumina sequencer or quantified by suitable procedures such as qPCR.For example, see Gong et al., Bioconjugate Chem. 2016, 27, 1, 217-225 for exemplary hybridization, extension and sequencing-based detection procedures.

[0387] The sequence reads generated by the sequencer are then analyzed using bioinformatics tools / algorithms. The molecular barcodes present in the sequenced molecules are used to identify binding molecules and their epitopes, while also deconvoluting the target proteins or PTMs they are in close proximity to. Quantification of the target protein (total protein) independent of PTMs is obtained from the reads corresponding to the binding of the second and fourth binding molecules. Quantification of the sequence reads corresponding to post-translationally modified proteins, for example, upregulated post-translationally modified proteins in tumor cells, in samples from patients compared to samples from healthy subjects facilitates determining the likelihood that the patient has cancer.

Claims

1. 1. A method for analyzing post-translationally modified proteins in a sample, comprising: a) Pre-enrichment of post-translationally modified proteins, i) contacting the sample or sub-sample thereof with a plurality of lectins, the plurality of lectins comprising: (A) a first lectin that specifically binds to a first saccharide present in a post-translational modification (PTM) on one or more target proteins in the sample, thereby generating a first complex comprising the first lectin and the target proteins; and (B) a second lectin that specifically binds to a second saccharide present in a PTM on one or more target proteins, thereby generating a second complex comprising the second lectin and the target proteins; and ii) separating said first complex and said second complex from other components of said sample or sub-sample, thereby obtaining at least one pre-enriched sub-sample. Pre-enrichment, including; b) determining the presence or level of at least one of said post-translationally modified target proteins, i) contacting the at least one pre-enriched sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, wherein each of the first and second binding molecules comprises a label; and ii) detecting the labels of the first and second binding molecules. and A method comprising:

2. 10. The method of claim 1, comprising obtaining first and second pre-enriched sub-samples, said second pre-enriched sub-sample comprising said other component.

3. 1. A method for analyzing post-translationally modified proteins in a sample, comprising: a) Pre-enrichment of post-translationally modified proteins, i) contacting the sample or sub-sample with a first lectin that specifically binds to a first saccharide present in a post-translational modification (PTM) on one or more target proteins in the sample, thereby producing a first complex comprising the first lectin and the target protein, wherein the first lectin is in solution during the contacting; and ii) separating said first complex from other components of said sample or sub-sample, thereby obtaining a first pre-enriched sub-sample. Pre-enrichment, including; b) determining the presence or level of at least one of said post-translationally modified target proteins, i) contacting the first pre-enriched sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, wherein each of the first and second binding molecules comprises a label; and ii) detecting the labels of the first and second binding molecules. and A method comprising:

4. 1. A method for analyzing post-translationally modified proteins in a sample, comprising: a) Pre-enrichment of post-translationally modified proteins, i) contacting the sample or sub-sample with a first lectin that specifically binds to a first saccharide present in a post-translational modification (PTM) on one or more target proteins in the sample, thereby generating a first complex comprising the first lectin and the target protein; and ii) separating said first complex from other components of said sample or sub-sample, thereby obtaining a first pre-enriched sub-sample comprising said first complex and a second sub-sample comprising said other components. Pre-enrichment, including; b) determining the presence or level of at least one of said post-translationally modified target proteins in at least said first pre-enriched sub-sample, and determining the presence or level of at least one target protein or post-translationally modified target protein in said second sub-sample, i) contacting the first pre-enriched sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, wherein the first and second binding molecules each comprise a label; and contacting the second sub-sample with at least one of the first binding molecule and the second binding molecule; and ii) detecting the labels of the first and second binding molecules bound to the first target protein in the first pre-enriched sub-sample, and detecting at least one of the labels of the first and second binding molecules bound to the first target protein in the second sub-sample. and A method comprising:

5. 1. A method for analyzing post-translationally modified proteins in a sample, comprising: a) Pre-enrichment of post-translationally modified proteins, i) contacting the sample or sub-sample with a first lectin that specifically binds to a first saccharide present in a post-translational modification (PTM) on one or more target proteins in the sample, thereby generating a first complex comprising the first lectin and the target protein; and ii) separating said first complex from other components of said sample or sub-sample, thereby obtaining a first pre-enriched sub-sample and a second sub-sample containing said other components. Pre-enrichment, including; b) determining the presence or level of at least one of said post-translationally modified target proteins in at least said first pre-enriched sub-sample, and determining the presence or level of at least one target protein or post-translationally modified target protein in said second sub-sample, i) contacting the first pre-enriched sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, wherein the first and second binding molecules each comprise a label; and contacting the second sub-sample with at least a third binding molecule that binds to a third epitope of the target protein that is different from the first and second epitopes, wherein the third binding molecule comprises a label; and ii) detecting the labels of the first and second binding molecules bound to the first target protein in the first pre-enriched sub-sample, and detecting the label of the third binding molecule bound to the third epitope in the second sub-sample. and A method comprising:

6. 6. The method of any one of claims 3 to 5, wherein the pre-enriching comprises contacting the sample or one or more sub-samples thereof with a plurality of lectins, the plurality of lectins comprising the first lectin and a second lectin that specifically binds to a second saccharide present in a PTM for one or more target proteins, wherein a second complex comprising the second lectin and the target protein is generated; and separating the first and second complexes from other components of the sample or one or more sub-samples thereof, thereby obtaining first and second pre-enriched sub-samples.

7. 4. The method of claim 1, wherein said separating said first complex from other components of said sample or sub-sample thereof further comprises obtaining a second sub-sample comprising said other components.

8. step (b) determining the presence or level of at least one of said post-translationally modified target proteins in at least said first pre-enriched sub-sample, and determining the presence or level of at least one target protein or post-translationally modified target protein in said second sub-sample; i) contacting the at least one pre-enriched sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, wherein the first and second binding molecules each comprise a label; and contacting the second sub-sample with at least one of the first binding molecule and the second binding molecule; and ii) detecting the labels of the first and second binding molecules bound to the first target protein in the first pre-enriched sub-sample, and detecting at least one of the labels of the first and second binding molecules bound to the first target protein in the second sub-sample.

10. The method of claim 9, comprising the steps of:

9. step (b) determining the presence or level of at least one of said post-translationally modified target proteins in at least said first pre-enriched sub-sample, and determining the presence or level of at least one target protein or post-translationally modified target protein in said second sub-sample; i) contacting the at least one pre-enriched sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, wherein the first and second binding molecules each comprise a label; and contacting the second sub-sample with at least a third binding molecule that binds to a third epitope of the target protein that is different from the first and second epitopes, wherein the third binding molecule comprises a label; and ii) detecting the labels of the first and second binding molecules bound to the first target protein in the first pre-enriched sub-sample, and detecting the label of the third binding molecule bound to the third epitope in the second sub-sample. The method of claim 7 , comprising the steps of:

10. 10. The method of any one of claims 1-2 or 4-9, wherein the first lectin or the plurality of lectins is in solution at the time of said contacting.

11. 1. A method for analyzing post-translationally modified proteins in a sample, comprising: a) Pre-enrichment of post-translationally modified proteins, i) contacting the sample or sub-sample thereof with a plurality of lectins, the plurality of lectins comprising: (A) a first lectin that specifically binds to a first saccharide present in a post-translational modification (PTM) on one or more target proteins in the sample, thereby forming a first complex comprising the first lectin and the target protein; and (B) a second lectin that specifically binds to a second saccharide present in a PTM on one or more target proteins, thereby forming a second complex comprising the second lectin and the target protein, wherein the plurality of lectins are in solution during the contacting, and wherein at least the first and second lectins each comprise a label that comprises an oligonucleotide; and ii) separating said first complex and said second complex from other components of said sample or sub-sample, thereby obtaining at least one pre-enriched sub-sample. Pre-enrichment, including; b) determining the presence or level of at least one of said post-translationally modified target proteins, i) contacting the at least one pre-enriched sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein and a second binding molecule that specifically binds to a second epitope of the first target protein, wherein the first and second binding molecules are antibodies, and each of the first and second binding molecules comprises a label; and ii) detecting the labels of the first and second binding molecules. and A method comprising:

12. The method of any one of claims 4 to 11, wherein the second sub-sample is a flow-through or a supernatant.

13. 13. The method of claims 1, 2, or 6-12, wherein each of the lectins in the plurality of lectins specifically binds to a different saccharide.

14. 14. The method of any one of claims 2 to 13, wherein the pre-enrichment comprises parallel pre-enrichment comprising contacting a first sub-sample of the sample with the first lectin and contacting a second sub-sample of the sample with the second lectin.

15. 14. The method of any one of claims 2 to 13, wherein the pre-enrichment comprises sequential pre-enrichment comprising contacting the sample or sub-sample thereof with the first lectin, separating the first complex from other components of the sample or sub-sample, thereby obtaining the first pre-enriched sub-sample and a first flow-through sub-sample comprising the other components of the sample or sub-sample, contacting the first flow-through sub-sample with the second lectin, and separating the second complex from other components of the first flow-through sub-sample, thereby obtaining the second pre-enriched sub-sample.

16. 2. The method of claim 1, wherein the pre-enrichment comprises simultaneously contacting the sample or sub-sample with multiple lectins, each lectin specifically binding to a different saccharide, the multiple lectins including the first lectin and a second lectin that specifically binds to a second saccharide present in a PTM for one or more target proteins, such that a second complex comprising the second lectin and the target protein is generated; and separating the first and second complexes from other components of the sample or one or more sub-samples thereof, thereby obtaining at least a first pre-enriched sub-sample.

17. 10. The method of any one of the preceding claims, wherein the first epitope does not comprise a PTM or a portion of a PTM.

18. The method of any one of claims 1 to 16, wherein the first epitope comprises a PTM or a portion of a PTM.

19. 10. The method of claim 1, wherein the first epitope does not include the first saccharide or a portion of the first saccharide.

20. 17. The method of any one of claims 1 to 16, wherein the first epitope does not comprise the first saccharide, a portion of the first saccharide, the second saccharide, or a portion of the second saccharide.

21. 21. The method of any one of claims 18 to 20, wherein the PTM or portion thereof of the first epitope comprises a saccharide, a phosphate moiety, a methyl moiety, an acetyl moiety, ubiquitin, a sumo moiety, a hydroxyl moiety, a lipid, or a nucleoside.

22. 10. The method of claim 1, wherein the PTM or portion thereof of the first epitope comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide.

23. 10. The method of claim 1, wherein the PTM or portion thereof of the first epitope comprises a monosaccharide, and optionally the monosaccharide is a GalNAc or Tn antigen.

24. 23. The method of claim 22, wherein the PTM of the portion of the first epitope comprises a tetrasaccharide, and optionally the tetrasaccharide is a sialyl Lewis saccharide.

25. 22. The method of claim 21 , wherein the PTM or portion thereof of the first epitope comprises a methyl moiety.

26. 10. The method of claim 1, wherein the first target protein is a histone.

27. 10. The method of any one of the preceding claims, wherein the second epitope does not comprise a PTM or a portion of a PTM.

28. The method of any one of claims 1 to 26, wherein the second epitope comprises a PTM or a portion of a PTM.

29. 10. The method of claim 1, wherein the second epitope does not comprise the first saccharide or a portion of the first saccharide.

30. 27. The method of any one of claims 2 to 26, wherein the second epitope does not comprise the first saccharide, a portion of the first saccharide, the second saccharide, or a portion of the second saccharide.

31. 31. The method of any one of claims 28-30, wherein the PTM or portion thereof of the second epitope comprises a saccharide, a phosphate moiety, a methyl moiety, an acetyl moiety, ubiquitin, a sumo moiety, a hydroxyl moiety, a lipid, or a nucleoside.

32. 10. The method of claim 9, wherein the PTM or portion thereof of the second epitope comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide.

33. 10. The method of claim 1, wherein the PTM or portion thereof of the second epitope comprises a monosaccharide, and optionally the monosaccharide is a GalNAc or Tn antigen.

34. 33. The method of claim 32, wherein the PTM or portion thereof of the second epitope comprises a tetrasaccharide, and optionally the tetrasaccharide is a sialyl Lewis saccharide.

35. 32. The method of claim 31 , wherein the PTM or portion thereof of the second epitope comprises a methyl moiety.

36. 10. The method of claim 1, wherein the first target protein is a histone.

37. 10. The method of claim 1, wherein the plurality of binding molecules comprises a third binding molecule that specifically binds to a third epitope of the first target protein, the third binding molecule comprising a label, and the detecting comprises detecting the label of the third binding molecule.

38. The method of claim 1, wherein the third epitope comprises a PTM or a portion of a PTM.

39. 39. The method of claim 37 or 38, wherein the third epitope does not include the first saccharide or a portion of the first saccharide.

40. 40. The method of any one of claims 37 to 39, wherein the third epitope does not comprise a saccharide that undergoes specific binding by the lectin used in the pre-enrichment step.

41. 41. The method of any one of claims 38-40, wherein the PTM or portion thereof of the third epitope comprises a saccharide, a phosphate moiety, a methyl moiety, an acetyl moiety, ubiquitin, a sumo moiety, a hydroxyl moiety, a lipid, or a nucleoside.

42. 10. The method of claim 9, wherein the PTM or portion thereof of the third epitope comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide.

43. 10. The method of claim 1, wherein the PTM or portion thereof of the third epitope comprises a monosaccharide, and optionally the monosaccharide is a GalNAc or Tn antigen.

44. 43. The method of claim 42, wherein the PTM or portion thereof of the third epitope comprises a tetrasaccharide, and optionally the tetrasaccharide is a sialyl Lewis saccharide.

45. 42. The method of claim 41 , wherein the PTM or portion thereof of the third epitope comprises a methyl moiety.

46. The method of claim 1, wherein the third epitope is an epitope on a histone target protein.

47. 47. The method of any one of claims 37-46, wherein the plurality of binding molecules comprises a fourth binding molecule that specifically binds to a fourth epitope of the first target protein, the fourth binding molecule comprising a label, and wherein the detecting comprises detecting the label of the fourth binding molecule.

48. The method of claim 1, wherein the fourth epitope comprises a PTM or a portion of a PTM.

49. 49. The method of claim 47 or 48, wherein the fourth epitope does not include the first saccharide or a portion of the first saccharide.

50. 50. The method of any one of claims 47 to 49, wherein the fourth epitope does not comprise a saccharide that undergoes specific binding by the lectin used in the pre-enrichment step.

51. 51. The method of any one of claims 47-50, wherein the PTM or portion thereof of the fourth epitope comprises a saccharide, a phosphate moiety, a methyl moiety, an acetyl moiety, ubiquitin, a sumo moiety, a hydroxyl moiety, a lipid, or a nucleoside.

52. 10. The method of claim 9, wherein the PTM or portion thereof of the fourth epitope comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide.

53. 10. The method of claim 1, wherein the PTM or portion thereof of the fourth epitope comprises a monosaccharide, and optionally the monosaccharide is a GalNAc or Tn antigen.

54. 53. The method of claim 52, wherein the PTM or portion thereof of the fourth epitope comprises a tetrasaccharide, and optionally the tetrasaccharide is a sialyl Lewis saccharide.

55. 52. The method of claim 51 , wherein the PTM or portion thereof of the fourth epitope comprises a methyl moiety.

56. The method of claim 1, wherein the fourth epitope is an epitope on a histone target protein.

57. 10. The method of claim 1, wherein the plurality of binding molecules comprises a binding molecule comprising a label that specifically binds to an epitope of a second target protein that does not comprise a PTM or a portion thereof, the second target protein comprising a PTM, and wherein the detecting comprises detecting the label of the at least one binding molecule that specifically binds to the second target protein.

58. 10. The method of claim 1, wherein the second target protein comprises a PTM that is specifically bound by the first, second, third, or fourth binding molecule.

59. 59. The method of claim 57 or 58, wherein the plurality of binding molecules comprises at least one binding molecule that binds to an epitope of the second protein that comprises a PTM or a portion thereof.

60. 10. The method of claim 1, further comprising separating each lectin from each associated target protein of each of the complexes prior to said contacting with said plurality of binding molecules.

61. 10. The method of claim 1, wherein the detection of the labels of the first and second binding molecules is used to quantify the first target protein in the sample or a sub-sample thereof.

62. a first sub-sample of said sample is contacted with said first lectin, said method comprising: contacting an input sub-sample of the sample with a second plurality of binding molecules comprising the first binding molecule and the second binding molecule; and detecting the labels of the first and second binding molecules bound to the first target protein in the input subsample.

10. The method of any one of the preceding claims, further comprising:

63. 10. The method of claim 1, wherein the detection of the labels of the first and second binding molecules bound to the first target protein in the input sub-sample is used to quantify the first target protein in the input sub-sample.

64. 64. The method of claim 62 or 63, wherein each of a plurality of target proteins is detected in the first sub-sample and in the input sub-sample using a plurality of labeled binding molecules specific for each of the plurality of target proteins.

65. 64. The method of claim 62 or 63, wherein each of a plurality of target proteins is quantified in the first sub-sample and in the input sub-sample using a plurality of labeled binding molecules specific for each of the plurality of target proteins.

66. 1. A method for analyzing post-translationally modified proteins in a sample, comprising: a) contacting the sample or sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein, a second binding molecule that specifically binds to a second epitope of the first target protein, and a third binding molecule that specifically binds to a third epitope, wherein the third epitope is an epitope of the first target protein or an epitope of a second target protein, the first epitope comprises a PTM or a portion of a PTM that comprises a saccharide, each of the first, second, and third binding molecules comprises a label, and each of the first, second, and third binding molecules comprises a lectin that specifically binds to the PTM; b) detecting the labels of the first, second, and third binding molecules; A method comprising:

67. 1. A method for analyzing post-translationally modified proteins in a sample, comprising: a) contacting the sample or sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein, a second binding molecule that specifically binds to a second epitope of the first target protein, and a third binding molecule that specifically binds to a third epitope, wherein the third epitope is an epitope of the first target protein or an epitope of a second target protein, the first epitope comprises a PTM or a portion of a PTM that comprises a saccharide, each of the first, second, and third binding molecules comprises a label, the first binding molecule comprises a lectin that specifically binds to the PTM, and the first lectin is in solution during the contacting; b) detecting the labels of the first, second, and third binding molecules; A method comprising:

68. contacting the sample or sub-sample thereof with the plurality of binding molecules produces a first set of complexes comprising the first binding molecule and the first target protein, the second binding molecule and the first target protein, and the third binding molecule and either the first target protein or the second target protein; a) separating said first set of complexes from other components of said sample or sub-sample, thereby producing a first sub-sample comprising said first set of complexes and a second sub-sample comprising said other components; b) contacting said second sub-sample with one or more binding molecules, including a fourth binding molecule that comprises a label and that specifically binds to a fourth epitope; c) detecting the label of the fourth binding molecule bound to the fourth epitope in the second sub-sample; 68. The method of claim 66 or claim 67, further comprising:

69. 1. A method for analyzing post-translationally modified proteins in a sample, comprising: a) contacting the sample or sub-sample with a plurality of binding molecules comprising a first binding molecule that specifically binds to a first epitope of a first target protein, a second binding molecule that specifically binds to a second epitope of the first target protein, and a third binding molecule that specifically binds to a third epitope, wherein the third epitope is an epitope of the first target protein or an epitope of a second target protein, and the first epitope is a PTM or PT comprising a saccharide. M, wherein each of the first, second, and third binding molecules comprises a label, and the first binding molecule comprises a lectin that specifically binds to the PTM, and wherein the step of contacting the sample or sub-sample with the plurality of binding molecules produces a first set of complexes comprising the first binding molecule and the first target protein, the second binding molecule and the first target protein, and the third binding molecule and either the first target protein or the second target protein; a) separating said first set of complexes from other components of said sample or sub-sample, thereby producing a first sub-sample comprising said first set of complexes and a second sub-sample comprising said other components; b) contacting the second sub-sample with one or more binding molecules, the binding molecule comprising a label and a fourth binding molecule that specifically binds to a fourth epitope; c) detecting the labels of the first, second, and third binding molecules in the first sub-sample and detecting the label of the fourth binding molecule in the second sub-sample; A method comprising:

70. 70. The method of claim 68 or 69, wherein the fourth epitope is an epitope of the first target protein.

71. 70. The method of claim 68 or 69, wherein the fourth epitope is an epitope of the second target protein.

72. 70. The method of claim 68 or 69, wherein the fourth epitope is an epitope of a third target protein.

73. 73. The method of any one of claims 68-72, wherein the second sub-sample is contacted with a plurality of binding molecules comprising the fourth binding molecule and a fifth binding molecule, wherein the fifth binding molecule specifically binds to a fifth epitope.

74. 74. The method of claim 73, wherein the fifth epitope is an epitope of the first target protein.

75. 74. The method of claim 73, wherein the fifth epitope is an epitope of the second target protein.

76. 74. The method of claim 73, wherein the fifth epitope is an epitope of a third target protein.

77. 74. The method of claim 73, wherein the fourth epitope is an epitope of a third target protein and the fifth epitope is an epitope of a fourth target protein.

78. 78. The method of any one of claims 66-77, wherein the third epitope is an epitope of the first target protein and comprises a PTM or a portion of a PTM other than the saccharide or portion thereof that is specifically bound by the first epitope.

79. 79. The method of any one of claims 61-78, wherein the third epitope is an epitope of the second target protein, the plurality of binding molecules comprises a fourth binding molecule that specifically binds to a fourth epitope, the fourth epitope is an epitope of the second target protein, and the third epitope comprises a PTM or a portion of a PTM.

80. 81. The method of any one of claims 66 to 80, wherein the second epitope does not comprise a PTM.

81. 81. The method of claim 79 or 80, wherein the fourth epitope does not include a PTM.

82. 82. The method of any one of claims 66-81, wherein each PTM or portion thereof that is specifically bound by one of the plurality of binding molecules is independently selected from a saccharide, a phosphate moiety, a methyl moiety, an acetyl moiety, ubiquitin, a sumo moiety, a hydroxyl moiety, a lipid, or a nucleoside.

83. 10. The method of claim 1, wherein at least one PTM or portion thereof comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide.

84. 10. The method of claim 1, wherein at least one PTM or portion thereof comprises a monosaccharide, optionally wherein said monosaccharide is a GalNAc or Tn antigen.

85. 84. The method of claim 83, wherein at least one PTM or portion thereof comprises a tetrasaccharide, and optionally the tetrasaccharide is a sialyl Lewis saccharide.

86. 83. The method of claim 82, wherein at least one PTM or portion thereof comprises a methyl moiety.

87. 10. The method of claim 1, wherein at least one target protein is a histone.

88. 10. The method of any one of the preceding claims, wherein the lectin, or at least one of the lectins, specifically binds to a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide.

89. 10. The method of claim 9, wherein the lectin, or at least one of the lectins, specifically binds to a monosaccharide, optionally wherein the monosaccharide is a GalNAc or Tn antigen.

90. 89. The method of claim 88, wherein the lectin, or at least one of the lectins, specifically binds to a tetrasaccharide, and optionally the tetrasaccharide is a sialyl Lewis saccharide.

91. 10. The method of claim 1, wherein at least one lectin and / or at least one binding molecule of the plurality of binding molecules is conjugated to a solid support.

92. 10. The method of claim 1, wherein the solid support comprises beads.

93. 10. The method of claim 1, wherein the solid support comprises magnetic beads.

94. 94. The method of any one of claims 91 to 93, wherein the first lectin is associated with the solid support.

95. 10. The method of claim 1, wherein each label independently comprises a fluorophore, biotin, a peptide, or an oligonucleotide.

96. 10. The method of claim 1, wherein each label comprises an oligonucleotide.

97. 10. The method of any one of the preceding claims, wherein the detection comprises a proximity ligation assay.

98. 97. The method of any one of claims 1 to 96, wherein said detecting comprises a proximity extension assay.

99. 99. The method of any one of claims 96 to 98, wherein each of the oligonucleotides of the label of each binding molecule that specifically binds to an epitope of the first target protein comprises a sequence that is complementary to a sequence of the label of at least one other binding molecule that specifically binds to an epitope of the first target protein.

100. 100. The method of any one of claims 96 to 99, wherein the oligonucleotides of the labels of the first and second binding molecules comprise sequences that are complementary to each other.

101. 101. The method of any one of claims 96 to 100, wherein the oligonucleotide of each label comprises an adaptor.

102. 10. The method of claim 1, wherein each adapter comprises a barcode.

103. 103. The method of any one of claims 96 to 102, wherein said detecting comprises amplifying said labeled oligonucleotides which are hybridized to each other.

104. 104. The method of claim 103, wherein said amplifying comprises quantitative amplification, optionally wherein said quantitative amplification comprises qPCR.

105. 105. The method of claim 103 or 104, wherein said detecting comprises sequencing said amplified oligonucleotides.

106. 106. The method of any one of claims 98 to 105, comprising a further amplification step after said proximity extension assay step, wherein a barcode is attached to said oligonucleotide label, said barcode corresponding to the type of PTM pre-enriched during said pre-enrichment step.

107. 107. The method of claim 106, wherein the barcode is a lectin type-specific barcode.

108. 97. The method of any one of claims 1 to 96, wherein said detecting comprises an immunoassay.

109. The method of the immediately preceding claim, wherein the immunoassay is an enzyme-linked immunosorbent assay, a sandwich assay, an electrochemiluminescence assay, or a multiplex immunoassay.

110. 97. The method of claim 95 or 96, wherein said detecting comprises flow cytometry analysis of said target protein.

111. 10. The method of any one of the preceding claims, comprising determining a level of one or more of the target proteins or one or more PTM-containing versions of the target proteins based on the detection.

112. 10. The method of any one of the preceding claims, wherein the sample is obtained from a subject.

113. 10. The method of claim 1, wherein the sample is a blood sample.

114. 10. The method of claim 1, wherein the blood sample is a whole blood sample.

115. 114. The method of claim 113, wherein the blood sample is a plasma sample.

116. 114. The method of claim 113, wherein the blood sample is a plasma pellet sample or a buffy coat sample.

117. 66. The method of any one of claims 1 to 65, wherein at least one binding molecule of the plurality of binding molecules comprises a protein.

118. 10. The method of claim 1, wherein at least one binding molecule of the plurality of binding molecules comprises a lectin other than any of the lectins used in the pre-enrichment step.

119. 10. The method of claim 1, wherein at least one binding molecule in the plurality of binding molecules comprises a protein other than a lectin.

120. The method of the immediately preceding claim, wherein at least one binding molecule of the plurality of binding molecules comprises VIM-1 or a methylcytosine-binding domain of VIM-1.

121. 10. The method of claim 1, wherein at least one binding molecule of the plurality of binding molecules comprises an antibody.

122. 10. The method of claim 1, wherein at least one binding molecule of the plurality of binding molecules comprises an aptamer.

123. 123. The method of any one of claims 1-122, wherein each of the plurality of binding molecules comprises a protein.

124. 10. The method of claim 9, wherein each of the plurality of binding molecules comprises an antibody.

125. 10. The method of any one of the preceding claims, wherein at least one target protein is a protein associated with a disease, or two or more of the plurality of target proteins are disease-associated molecules, or each of the plurality of target proteins is a protein associated with a disease.

126. The method of the preceding claim, wherein the disease is cancer.

127. 10. The method of claim 1, wherein the at least one target protein is differentially post-translationally modified in tumor cells compared to healthy cells of the same tissue type.

128. 10. The method of claim 9, wherein the at least one target protein is upregulated in tumor cells compared to healthy cells of the same tissue type.

129. 129. The method of any one of claims 125-128, wherein at least one, two or more, or each, of the target proteins is selected from RBl, TP53, PTEN, NF1, BRCA1, CEACAM1, CEACAM5, CEACAM6, EGFR, ErbB2, ErbB3, ErbB4, β-catenin, PD-Ll, CTLA4, NYESOL, mesothelin, CA15-3, CA19-9, CA-125, CA27-29, and CA-72-4.

130. 130. The method of any one of claims 125 to 129, wherein at least one target protein, two or more target proteins, or each of said plurality of target proteins is a cell type marker.

131. 10. The method of claim 8, wherein the cell type marker is selected from markers of immune cells and solid tissue cells.

132. 10. The method of claim 9, wherein the cell type marker is selected from colon, lung, breast, skin, prostate, stomach, pancreatic markers and liver cell type markers.

133. 10. The method of any one of the preceding claims, comprising analyzing DNA in a sub-sample of the sample or in a second sample obtained from the same subject from which the first sample is obtained.

134. 10. The method of claim 1, wherein the sub-sample or second sample is a plasma or serum sample.

135. 10. The method of claim 1, wherein the DNA is cfDNA.

Images