WO2023102502A4 - Enriched peptide detection by single molecule sequencing - Google Patents
Enriched peptide detection by single molecule sequencing Download PDFInfo
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- WO2023102502A4 WO2023102502A4 PCT/US2022/080781 US2022080781W WO2023102502A4 WO 2023102502 A4 WO2023102502 A4 WO 2023102502A4 US 2022080781 W US2022080781 W US 2022080781W WO 2023102502 A4 WO2023102502 A4 WO 2023102502A4
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims 73
- 238000001514 detection method Methods 0.000 title 1
- 238000012163 sequencing technique Methods 0.000 title 1
- 102000004169 proteins and genes Human genes 0.000 claims abstract 12
- 108090000623 proteins and genes Proteins 0.000 claims abstract 12
- 239000012472 biological sample Substances 0.000 claims abstract 10
- 108091034117 Oligonucleotide Proteins 0.000 claims 30
- 239000011230 binding agent Substances 0.000 claims 23
- 239000000523 sample Substances 0.000 claims 21
- 102000004196 processed proteins & peptides Human genes 0.000 claims 14
- 230000002797 proteolythic effect Effects 0.000 claims 11
- 125000003277 amino group Chemical group 0.000 claims 9
- 230000029087 digestion Effects 0.000 claims 9
- 238000000034 method Methods 0.000 claims 8
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 claims 7
- 238000007385 chemical modification Methods 0.000 claims 7
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims 6
- 239000004472 Lysine Substances 0.000 claims 6
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims 6
- 239000000203 mixture Substances 0.000 claims 4
- 239000002253 acid Substances 0.000 claims 3
- 150000001413 amino acids Chemical group 0.000 claims 3
- 230000000295 complement effect Effects 0.000 claims 3
- 208000035657 Abasia Diseases 0.000 claims 2
- 230000027455 binding Effects 0.000 claims 2
- 238000009396 hybridization Methods 0.000 claims 2
- 238000003780 insertion Methods 0.000 claims 2
- 125000000729 N-terminal amino-acid group Chemical group 0.000 claims 1
- 235000009074 Phytolacca americana Nutrition 0.000 claims 1
- 240000007643 Phytolacca americana Species 0.000 claims 1
- 238000003776 cleavage reaction Methods 0.000 claims 1
- 108020004707 nucleic acids Proteins 0.000 claims 1
- 102000039446 nucleic acids Human genes 0.000 claims 1
- 150000007523 nucleic acids Chemical class 0.000 claims 1
- 230000000379 polymerizing effect Effects 0.000 claims 1
- 230000007017 scission Effects 0.000 claims 1
- 230000009870 specific binding Effects 0.000 claims 1
- 108010067902 Peptide Library Proteins 0.000 abstract 1
- 239000008280 blood Substances 0.000 abstract 1
- 210000004369 blood Anatomy 0.000 abstract 1
- 238000005259 measurement Methods 0.000 abstract 1
- 230000035945 sensitivity Effects 0.000 abstract 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The inventions herein, e.g., relate to quantitative measurement of proteins, and provide significant improvements, e.g., in the sensitivity, accuracy, throughput and cost of measuring clinically important proteins in biological samples such as blood. The inventions herein, e.g., also relate to peptide library preparation for quantitative single molecule analysis.
Claims
1. A stoichiometrically-flattened standardized sample, comprising a plurality of pairs of cognate standard and target peptide constructs enriched from a standardized proteolytic digest of a biological sample by binding to their respective cognate binders, wherein a pre-enrichment ratio calculated by dividing the number of molecules of a first target peptide construct that is the most numerous of said target peptide constructs in the standardized sample digest by die number of molecules of a second target peptide construct that is the least numerous of said target peptide constructs in the standardized sample digest is more than 10 times larger than a post-enrichment ratio calculated by dividing the number of molecules of said first target peptide construct by the number of molecules of said second target peptide construct in said enriched sample.
2. A mixture of a plurality of BINDERS cogna te to a plurality of pairs of cognate STANDARD and TARGET peptide constructs, wherein said mixture lias the capacity to bind and thereby enrich different relative amounts of different TARGET peptide constructs such that a preenrichment ratio calculated by dividing die number of molecules of a first target peptide construct that is the most numerous of said target peptide constructs in the standardized sample digest by the number of molecules of a second target peptide construct that is the least numerous of said target peptide constructs in the standardized sample digest is more than 10 times larger than a post-enrichment ratio calculated by dividing the number of molecules of said first target peptide construct by the number of molecules of said second target peptide construct in said enriched sample.
3. A set of cognate molecules comprising
(a) a TARGET construct comprising a TARGET peptide generated by proteolytic digestion of a target protein in a biological sample and an oligonucleotide TARGET tag.
(b) a STANDARD construct comprising a synthetic STANDARD peptide comprising at least 50% of the amino acid sequence of said TARGET peptide and an oligonucleotide tag having a sequence different from said TARGET tag, and
(c) a BINDER capable of binding a mixture of said TARGET constructs and said STANDARD constructs wherein the ratio of said TARGET’’ and STANDARD constructs bound bv said BINDER differs from the ratio of said TARGET and STAND ARD constructs in said mixture by no more than 20%.
4. A standardized sample digest derived from a proteolytic digest of a biological sample, comprising: an amount of a molecular construct comprising a TARGET tag and a TARGET peptide, said construct being a TARGET peptide construct and an amount of a molecular construct comprising a STANDARD tag and a peptide whose sequence is the same or similar to the sequence of said TARGET peptide, said construct being a STANDARD peptide construct, wherein the TARGET peptide is generated by proteolytic digestion of a target protein in said biological sample, wherein said TARGET and STANDARD tags can be distinguished by a single molecule detector and comprise oligonucleotides covalently joined to peptides in their respective constructs, wherein said TARGET tag is covalently attached to a plurality of the peptides present in said sample digest, wherein said TARGET peptide construct comprises more than 90% of the target peptide molecules present in said sample digest and wherein said STANDARD peptide construct is prepared separately and added to said digest in a known amount, or in a consistent relative amount across a multiplicity of samples
5. The standardized sample digest of claim 4, wherein the number of molecules of the standard peptide construct added to the sample digest differs by no more than a factor of 100 from the number of molecules of the target peptide construct in said sample digest.
6. An enriched standardized sample digest derived from a proteolytic digest of a biological sample, comprising:
an amount of a molecular construct comprising a TARGET tag and a TARGET peptide, said construct being a TARGET peptide construct and an amount of a molecular construct comprising a STANDARD tag and a peptide whose sequence is the same or similar to the sequence of said TARGET peptide, said construct being a STANDARD peptide construct, wherein the TARGET peptide is generated by proteolytic digestion of a target protein in said biological sample, wherein said TARGET and STANDARD tags can be distinguished by a single molecule detector and comprise oligonucleotides covalently joined to peptides in their respective constructs, wherein said TARGET tag is covalently attached to a plurality of the peptides present in said sample digest, wherein said TARGET peptide construct comprises more than 90% of the target peptide molecules present in said sample digest, wherein said STANDARD peptide construct is prepared separately and added to said digest in a known amount, or in a consistent relative amount across a multiplicity7 of samples, and wherein said TARGET and STANDARD peptide constructs are bound to a cognate BINDER in a ratio equal within 2%, 5%, 10% or 20% to the ratio in which they are present in said standardized sample digest.
7. A method for the measuring the amounts of first and second target proteins present in a biological sample at molar abundances differing by more than a factor of 100, comprising: proteolytically digesting said sample, modifying a plurality'7 of peptides in the digested sample by adding a target tag to form a plurality of constructs comprising selected first and second I'ARGET peptides derived from, and proteotypic of, said first and second target proteins, said plurality of constructs being TARGET construct molecules, adding an amount that is known and/or consistent between a set of samples of prepared first and second STANDARD peptide constructs that are cognates respectively of said first and
second TARGET peptide constructs and comprise a STANDARD tag, forming a standardized digest, enriching said cognate T ARGET and STANDARD peptide constructs by contacting said standardized digest with first and second cognate BINDERS, forming bound constructs, separating said bound constructs from unbound constructs to form enriched constructs, optionally releasing said enriched constructs from said BINDERS, counting said enriched first and second TARGET construct molecules and said enriched first and second STANDARD construct molecules using a sequence-sensitive single molecule detector capable of distinguishing said TARGET and STANDARD tags and identifying said first and second peptides, and calculating the amount of said first protein in said sample using the ratio of the number of said enriched first TARGET construct molecules to said enriched first STANDARD construct molecules multiplied by the known amount of said first STANDARD construct added, and calculating the amount of said second protein in said sample using the ratio of the number of said enriched second TARGET construct molecules to said enriched second STANDARD construct molecules multiplied by the known amount of said second STANDARD construct added, wherein the numbers of said first and said second enriched TARGET constructs differ by less than a factor of 10 after enrichment.
8. A method of encoding in a TARGET' peptide:oligonucleotide construct the identity of a cognate BINDER to which said construct is bound, consisting of the steps of
(a) specific binding of the peptide portion of said construct to said cognate BINDER, wherein said BINDER comprises an oligonucleotide BINDER tag whose sequence identifies said BINDER,
(b) hybridization of said BINDER tag, or an adapter comprising an oligonucleotide sequence complementary to said BINDER tag, with an oligonucleotide sequence present in said TARGET construct,
(c) addition of an oligonucleotide BINDER_ID tag sequence identifying said BINDER to said TARGET construct, by either (1 ) hybridization of said BINDER ID tag with said BINDER tag, or an assembler adapter comprising an oligonucleotide sequence complementary to said BINDER tag, forming a nucleic acid duplex wherein an end of said BINDER ID tag is located adjacent to an end of said oligonucleotide sequence present in said TARGET construct, and effecting a ligation of said TARGET construct and said BINDER ID tag or (2) polymerizing an extension to said 'TARGET construct sequence directed by said BINDER tag, or an adapter comprising an oligonucleotide sequence complementary to said BINDER tag,
(d) dissociating said extended GARGET construct from said BINDER.
9. 'rhe method of claim 7, wherein said construct molecules are joined together into concatamers prior to presentation to said detector.
10. A method for preparation of a TARGET construct comprising the steps of:
(a) a first proteolytic digestion of proteins in a biological sample yielding a plurality of peptides each comprising a single lysine residue,
(b) a first chemical modifica tion of peptide amino groups in said digest, including alpha ammo groups and lysine epsilon ammo groups, introducing a linkage to a first non-peptidic construct component,
(c) a second proteolytic digestion cleaving said peptides comprising said first chemical modification at one or more sites not cleaved in said first digestion, yielding a second plurality of peptides comprising a chemically modified lysine epsilon ammo group and an unmodified alpha ammo group,
(d) a second chemical modification of said unmodified alpha amino groups introducing a linkage to a second non-peptidic construct component.
11. The method of 10 wherein said first proteolytic digestion yields a plurality of peptides having a c-terminal lysine ammo acid, and wherein said second proteolytic digestion cleaves peptides either n-termmal or c-terminal to an amino acid other than lysine, resulting in a plurality
of linear constructs comprising peptides having said first chemical modification on said lysine epsilon amino groups and second chemical modification on said n-tenninal amino groups.
12. A method for preparation of a TARGET construct comprising the steps of
(a) a first chemical modification of amino groups of proteins in a biological sample, including alpha amino groups and lysine epsilon ammo groups, introducing a linkage to a first non-peptidic construct component,
(b) a proteolytic digestion of said proteins yielding a plurality of peptides comprising a chemically modified lysine epsilon amino group and an unmodified alpha amino group
(c) a second chemical modification of said unmodified alpha amino groups introducing a linkage to a second nompeptidic construct component different from said first nompeptidic construct component.
13. The method of claims 10 or 12 wherein ei t her or both of said first and second non-peptidic construct components comprise respective first and second oligonucleotides or are used subsequently to establish a linkage with a first and second oligonucleotide, said first and second oligonucleotides having different sequences.
14. A linear TARGET construct comprising a peptide and a first and a second oligonucleotide, wherein said first oligonucleotide is chemically linked to the epsilon ammo group of a c-terminal lysine residue of the TARGET peptide and said second oligonucleotide is linked to the n- terrmnal ammo acid of the TARGET peptide.
15. A linear TARGET construct of 14 wherein a) said first and second oligonucleotides have different sequences and wherein either b) the 5‘ end of said first oligonucleotide is chemically linked to the epsilon ammo group of a c-terminal lysine residue of the TARGET peptide and the 3' end of said second oligonucleotide is linked to the n-terminal ammo acid of the TARGET peptide, or c) the 3' end of said first oligonucleotide is chemically linked to the epsilon ammo group of a c-terminal lysine residue of the TARGET peptide and the 5' end of said second oligonucleotide is linked to the n-terminal amino acid of the TARGET peptide.
16. A rope-tow TARGET construct comprising an oligonucleotide and a peptide, wherein one of the terminal amino acids of said peptide is linked to a site in said oligonucleotide adjacent to or within an abasic stretch in said oligonucleotide, said abasic stretch having a length greater than
80% of the length of said peptide in an extended configuration.
17. A loop-insertion TARGET construct comprising an oligonucleotide comprising a first site and a second site, said sites respectively covalently linked to the n-tenninus and the side chain of a c-terminal lysine of a TARGET peptide, forming a loop structure.
18. A method of generating a linear TARGET construct by clea vage of the oligonucleotide of the loop-insertion TARGET construct of claim 17 at a site between said first and second sites. yielding a construct comprising a peptide and first and second oligonucleotides, said oligonucleotides respectively comprising said first and second sites, attached to the c-terminal and n-terminal ends of said peptide.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP22902411.2A EP4441277A2 (en) | 2021-12-01 | 2022-12-01 | Enriched peptide detection by single molecule sequencing |
| CA3238472A CA3238472A1 (en) | 2021-12-01 | 2022-12-01 | Enriched peptide detection by single molecule sequencing |
| AU2022400954A AU2022400954A1 (en) | 2021-12-01 | 2022-12-01 | Enriched peptide detection by single molecule sequencing |
Applications Claiming Priority (18)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US202163284990P | 2021-12-01 | 2021-12-01 | |
| US63/284,990 | 2021-12-01 | ||
| US202163288987P | 2021-12-13 | 2021-12-13 | |
| US63/288,987 | 2021-12-13 | ||
| US202263296196P | 2022-01-04 | 2022-01-04 | |
| US63/296,196 | 2022-01-04 | ||
| US202263303417P | 2022-01-26 | 2022-01-26 | |
| US63/303,417 | 2022-01-26 | ||
| US202263313760P | 2022-02-25 | 2022-02-25 | |
| US63/313,760 | 2022-02-25 | ||
| US202263340001P | 2022-05-10 | 2022-05-10 | |
| US63/340,001 | 2022-05-10 | ||
| US202263348213P | 2022-06-02 | 2022-06-02 | |
| US63/348,213 | 2022-06-02 | ||
| US202263352925P | 2022-06-16 | 2022-06-16 | |
| US63/352,925 | 2022-06-16 | ||
| US202263373875P | 2022-08-30 | 2022-08-30 | |
| US63/373,875 | 2022-08-30 |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| WO2023102502A2 WO2023102502A2 (en) | 2023-06-08 |
| WO2023102502A3 WO2023102502A3 (en) | 2023-09-14 |
| WO2023102502A4 true WO2023102502A4 (en) | 2023-10-12 |
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| Application Number | Title | Priority Date | Filing Date |
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| PCT/US2022/080781 WO2023102502A2 (en) | 2021-12-01 | 2022-12-01 | Enriched peptide detection by single molecule sequencing |
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| EP (1) | EP4441277A2 (en) |
| AU (1) | AU2022400954A1 (en) |
| CA (1) | CA3238472A1 (en) |
| WO (1) | WO2023102502A2 (en) |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050255491A1 (en) * | 2003-11-13 | 2005-11-17 | Lee Frank D | Small molecule and peptide arrays and uses thereof |
| AU2013284452B2 (en) * | 2012-06-27 | 2017-08-31 | Siscapa Assay Technologies, Inc. | Multipurpose mass spectrometric assay panels for peptides |
| JP6088710B2 (en) * | 2013-08-19 | 2017-03-01 | シンギュラー・バイオ・インコーポレイテッド | Single molecule detection assays and uses thereof |
| WO2019089846A1 (en) * | 2017-10-31 | 2019-05-09 | Encodia, Inc. | Methods and compositions for polypeptide analysis |
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2022
- 2022-12-01 EP EP22902411.2A patent/EP4441277A2/en active Pending
- 2022-12-01 CA CA3238472A patent/CA3238472A1/en active Pending
- 2022-12-01 AU AU2022400954A patent/AU2022400954A1/en active Pending
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| CA3238472A1 (en) | 2023-06-08 |
| AU2022400954A1 (en) | 2024-07-11 |
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| EP4441277A2 (en) | 2024-10-09 |
| WO2023102502A3 (en) | 2023-09-14 |
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