# US 2021/0239705 A1 · Methods and applications of protein identification > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2021/0239705 A1 - Document ID: US20210239705A1 - Status: Published application - Kind: A1 - Application number: 16/972,341 - Filed: 2019-06-05 - Published: 2021-08-05 - Earliest claimed: 2018-06-06 - Assignees: Nautilus Biotechnology, Inc. - Applicants: Nautilus Biotechnology, Inc. - Inventors: Parag MALLICK - Web page: https://patentgrep.com/patent/US20210239705A1 ## CPC - C40B 30/04: by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding - G01N 33/6842: {Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins} - G01N 33/54306: {Solid-phase reaction mechanisms} - G01N 33/582: {with fluorescent label} - G01N 33/6803: {General methods of protein analysis not limited to specific proteins or families of proteins} - C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract Methods and systems for identifying a protein within a sample are provided herein. A panel of antibodies are acquired, none of which are specific for a single protein or family of proteins. Additionally, the binding properties of the antibodies in the panel are determined. Further, the protein is iteratively exposed to a panel of antibodies. Additionally, a set of antibodies which bind the protein are determined. The identity of the protein is determined using one or more deconvolution methods based on the known binding properties of the antibodies to match the set of antibodies to a sequence of a protein. ## Claims ### Claim 1 A method of determining protein characteristics, the method comprising: obtaining a substrate in which portions of one or more proteins are conjugated to the substrate such that each individual (at the molecular level) protein portion has a unique, optically resolvable, spatial address; applying a fluid containing a first through (ordered) nth set of one or more affinity reagents to the substrate, wherein each of the one or more affinity reagents is specific to one epitope (contiguous or non-contiguous amino acid sequence) of a portion of the one or more proteins, and wherein each affinity reagent of the first through nth set of one or more of affinity reagents is linked to an identifiable tag; after each application to the substrate of the first and subsequent through nth set of one or more of affinity reagents, performing the following steps: observing the identifiable tag; identifying one or more unique spatial addresses of the substrate having one or more observed signal; determining that each portion of the one or more proteins having an identified unique spatial address contains the one or more epitopes associated with the one or more observed signals; and determining the characteristics of each protein portion based on the one or more epitopes. ### Claim 2 (depends on 1) The method of claim 1, wherein each affinity reagent of the first through nth set of one or more affinity reagents is not specific to an individual proteins or protein families, but is specific to a portion of the one or more individual distinguishable proteins. ### Claim 3 (depends on 1) The method of claim 1, wherein each affinity reagent of the first through nth set of one or more affinity reagents recognizes a family of one or more epitopes that are present in more than one proteins. ### Claim 4 (depends on 1) The method of claim 1, wherein the epitope of the portion of the one or more proteins is conformational or linear. ### Claim 5 (depends on 1) The method of claim 1, wherein the one or more affinity reagents comprises contiguous or non-contiguous amino acids that are specific to the corresponding epitopes. ### Claim 6 (depends on 5) The method of any one of claim 5, further comprising: determining the identity of the portion of the one or more proteins to a threshold degree of accuracy based on the determined one or more epitopes within the portion. ### Claim 7 The method of any one of the preceding claims, wherein the first through nth set of one or more affinity reagents comprises more than 100 affinity reagents. ### Claim 8 The method of any one of the preceding claims, wherein the method further comprises the use of affinity reagents which bind a single protein or single protein isoform. ### Claim 9 The method of any one of the preceding claims, further comprising: determining the identity of the portion of the one or more proteins to a threshold degree of accuracy based on the pattern of binding of the affinity reagents. ### Claim 10 The method of any one of the preceding claims, wherein the substrate is a flow cell. ### Claim 11 The method of any one of the preceding claims, wherein the portions of one or more proteins are conjugated to the substrate using a photo-activatable linker. ### Claim 12 The method of any one of the preceding claims, wherein the portions of one or more proteins are conjugated to the substrate using a photo-cleavable linker. ### Claim 13 The method of any one of the preceding claims, wherein at least a portion of the at least one set of affinity reagents is modified to be conjugated to an identifiable tag. ### Claim 14 The method of any one of the preceding claims, wherein an identifiable tag is a fluorescent tag. ### Claim 15 The method of any one of the preceding claims, wherein an identifiable tag is a nucleic acid barcode. ### Claim 16 The method of any one of any one of the preceding claims, wherein the number of spatial addresses occupied by an identified portion of a protein is counted to quantify the level of that protein in the sample. ### Claim 17 The method of any one of the preceding claims, wherein the identity of the portion of the one or more proteins is determined using deconvolution software. ### Claim 18 The method of any one of the preceding claims, wherein the identity of the portion of the one or more proteins is determined by decoding combinations of epitopes associated with unique spatial addresses. ### Claim 19 The method of any of the preceding claims, further comprising: denaturing the one or more proteins prior to conjugating the portions of the one or more proteins to the substrate. ### Claim 20 The method of any one of the preceding claims, wherein the portions of one or more proteins to a substrate are present in a complex mixture of multiple proteins. ### Claim 21 The method of any one of the preceding claims, wherein the method is used to identify multiple proteins. ### Claim 22 A method of identifying a protein comprising: acquiring a panel of antibodies none of which are specific for a single protein or family of proteins, determining the binding properties of the antibodies in the panel, iteratively exposing the protein to the panel of antibodies, determining a set of the antibodies which bind the protein, and using one or more deconvolution methods based on the known binding properties of the antibodies to match the set of antibodies to a sequence of a protein, thereby determining the identity of the protein. ### Claim 23 (depends on 22) The method of claim 22, wherein the protein to be identified is identified within a sample containing multiple different proteins. ### Claim 24 (depends on 22) The method of claim 22, wherein the method is able to simultaneously identify multiple proteins within a single sample. ### Claim 25 A method of identifying a protein comprising: acquiring a panel of antibodies none of which are specific for a single protein or family of proteins, determining the binding properties of the antibodies in the panel, iteratively exposing the protein to the panel of antibodies, determining a set of the antibodies which do not bind the protein, and using one or more deconvolution methods based on the known binding properties of the antibodies to match the set of antibodies to a sequence of a protein, thereby determining the identity of the protein. ### Claim 26 A method of uniquely identifying and quantifying n proteins in a mixture of proteins using m affinity reagents, wherein n is larger than m, and n and m are positive integers greater than 1, and wherein the proteins have not been separated by an intrinsic property. ### Claim 27 (depends on 26) The method of claim 26, wherein n is approximately 5 times larger than m. ### Claim 28 (depends on 26) The method of claim 26, wherein n is approximately 10 times larger than m. ### Claim 29 (depends on 26) The method of claim 26, wherein n is approximately 20 times larger than m. ### Claim 30 A method of uniquely identifying and quantifying n proteins in a mixture of proteins using m binding reagents, wherein n is larger than m, and wherein the proteins are randomly arranged. ### Claim 31 (depends on 26) The method of claim 26, wherein the proteins have not been separated by a size based, or charge based, separation method. ### Claim 32 A method of uniquely identifying and quantifying n single protein molecules in a mixture of protein molecules using m affinity reagents, wherein n is larger than m, and wherein the single protein molecules are conjugated to a substrate and spatially separated such that each individual protein molecule has a unique, optically resolvable, spatial address. ### Claim 33 A method to identify, with certainty above a threshold amount, an unknown single protein molecule from a pool of n possible proteins, using a panel of affinity reagents, wherein the number of affinity reagents in the panel is m, and wherein m is less than one tenth of n. ### Claim 34 A method to select a panel of m affinity reagents capable of identifying an unknown protein selected from a pool of n possible proteins, wherein m is less than n−1. ### Claim 35 A method to select a panel of m affinity reagents capable of identifying an unknown protein selected from a pool of n possible proteins, wherein m is less than one tenth of n. ### Claim 36 A method to select a panel of less than 4000 affinity reagents, such that the panel of less than 4000 affinity reagents is capable of uniquely identifying each of 20,000 different proteins. ### Claim 37 A method of uniquely identifying and quantifying n proteins in a mixture of proteins using m binding reagents, wherein m is less than n−1, and wherein each protein is identified via a unique profile of binding by a subset of the m the binding reagents. ### Claim 38 A method of uniquely identifying a single protein molecule, the method comprising: obtaining a panel of affinity reagents; exposing the single protein molecule to each of the affinity reagents in the panel; determining whether each affinity reagent binds or does not bind the single protein molecule; and using the collected binding data to determine the identity of the single protein molecule, wherein the identity of the single protein molecule cannot be determined by the binding data of any individual affinity reagent in the panel of affinity reagents. ### Claim 39 A method that is capable of identifying more than 20% of proteins in the human proteome from a protein sample, wherein the proteins are not substantially destroyed in the process. ### Claim 40 (depends on 39) The method of claim 39, wherein the method does not require more than 4000 affinity reagents. ### Claim 41 (depends on 39) The method of claim 39, wherein the method does not require more than 100 mg of the protein sample. ### Claim 42 A method of determining protein characteristics, the method comprising: obtaining a substrate with portions of one or more proteins conjugated to the substrate such that each individual protein portion has a unique, optically resolvable, spatial address; applying a fluid containing a set of affinity reagents, selected from 1-n sets of one or more affinity reagents, to the substrate; after each application of a set of one or more of affinity reagents to the substrate, performing the following steps: observing bound affinity reagents; identifying one or more unique spatial addresses of the substrate having one or more bound affinity reagents; and determining that each portion of the one or more proteins having an identified unique spatial address contains the one or more epitopes associated with the one or more observed signals.