Search

US-20260126449-A1 - POLYPEPTIDE CAPTURE, IN SITU FRAGMENTATION AND IDENTIFICATION

US20260126449A1US 20260126449 A1US20260126449 A1US 20260126449A1US-20260126449-A1

Abstract

The present disclosure provides a method of identifying a polypeptide. The method can include steps of (a) attaching a polypeptide to a particle or solid support, thereby producing an immobilized polypeptide having a plurality of amino acids linked to the particle or solid support; (b) fragmenting the immobilized polypeptide, whereby the particle is attached to a set of fragments of the polypeptide; (c) performing a binding assay including contacting the set of fragments with a plurality of affinity reagents and detecting binding of affinity reagents of the plurality of affinity reagents to the set of fragments; and (d) identifying the polypeptide from results of the binding assay

Inventors

  • Sujal M. Patel
  • Parag Mallick

Assignees

  • NAUTILUS SUBSIDIARY, INC.

Dates

Publication Date
20260507
Application Date
20251218

Claims (20)

  1. 1 . A method, comprising: (a) attaching a polypeptide to a nucleic acid particle, wherein the polypeptide includes a plurality of first attachment moieties, wherein the nucleic acid particle includes a first linker composed of double stranded nucleic acid and having a single second attachment moiety reactive with one of the plurality of the first attachment moieties of the polypeptide thereby immobilizing the polypeptide to the nucleic acid particle; (b) after attaching the polypeptide to the nucleic acid particle, modifying the nucleic acid particle by forming a plurality of second linkers on the nucleic acid particle, each of the second linkers composed of double stranded nucleic acid and each of the second linkers having a single second attachment moiety reactive with one of the plurality of first attachment moieties of the polypeptide; and (c) fragmenting the immobilized polypeptide, thereby creating a set of polypeptide fragments that are individually immobilized to the nucleic acid particle via the first linker or the second linkers.
  2. 2 . The method of claim 1 , wherein the set of polypeptide fragments includes a first fragment attached to the nucleic acid particle via the first linker, and two or more fragments attached to the nucleic acid particle via the second linkers.
  3. 3 . The method of claim 1 , further comprising: (d) performing a binding assay comprising contacting the set of polypeptide fragments with a plurality of affinity reagents and detecting binding of affinity reagents of the plurality of affinity reagents to the set of polypeptide fragments.
  4. 4 . The method of claim 3 , further comprising: (e) characterizing the polypeptide from results of the binding assay.
  5. 5 . The method of claim 4 , wherein characterizing includes identifying the polypeptide from the results of the binding assay.
  6. 6 . The method of claim 4 , wherein characterizing includes quantifying the polypeptide in a sample.
  7. 7 . The method of claim 1 , wherein the polypeptide is a full-length amino acid sequence.
  8. 8 . The method of claim 1 , wherein the polypeptide immobilized to the nucleic acid particle is the only polypeptide immobilized to the nucleic acid particle.
  9. 9 . The method of claim 1 , wherein the plurality of second linkers each have a first portion having double stranded nucleic acid and a second portion having single stranded nucleic acid.
  10. 10 . The method of claim 1 , wherein each of the plurality of second linkers are attached to the nucleic acid particle at predefined sites.
  11. 11 . The method of claim 10 , wherein the predefined sites correspond to single stranded nucleic acid regions of the nucleic acid particle.
  12. 12 . The method of claim 1 , wherein the plurality of second linkers each include: (i) a first portion having the double stranded nucleic acid, and (ii) a second portion having single stranded nucleic acid.
  13. 13 . The method of claim 1 , further comprising: (d) after attaching the polypeptide to the nucleic acid particle, modifying lengths of the plurality of second linkers.
  14. 14 . The method of claim 13 , wherein modifying lengths of the plurality of second linkers includes shortening the lengths of the plurality of second linkers.
  15. 15 . The method of claim 14 , wherein the plurality of second linkers each include: (i) a first portion having the double stranded nucleic acid, and (ii) a second portion having single stranded nucleic acid, and shortening the lengths of the plurality of second linkers includes hybridizing complementary nucleic acid strands to the second portions having the single stranded nucleic acid.
  16. 16 . The method of claim 1 , wherein the first linker includes a different nucleic acid sequence than the plurality of second linkers.
  17. 17 . The method of claim 16 , wherein the plurality of second linkers includes the same nucleic acid sequences.
  18. 18 . The method of claim 1 , wherein the first attachment moiety and the second attachment moieties are click moieties.
  19. 19 . The method of claim 1 , further comprising: (d) modifying the polypeptide by adding exogenous moieties at amino acids of the polypeptide as the first attachment moieties.
  20. 20 . The method of claim 1 , wherein the first attachment moieties are positioned at lysine amino acids of the polypeptide.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation application of U.S. application Ser. No. 18/463,063, filed Sep. 7, 2023, which claims priority to U.S. Provisional Application No. 63/377,249, filed on Sep. 27, 2022, each of which is incorporated herein by reference in their entirety. BACKGROUND Tools for characterizing and quantifying proteome dynamics with single-molecule resolution stand to overcome myriad challenges in biomedicine, including the measurement of low abundance polypeptides in therapeutic development, discovery of biomarkers for clinically actionable maladies, and diagnosing patients in clinical settings. Additionally, such tools could provide insights into the molecular heterogeneity of populations of proteoforms, which is currently masked by bulk measurements employed in research and clinical settings. Proteomes are large and diverse, having hundreds of thousands of different polypeptides and a wide dynamic range in the level of each type of polypeptide. The number and diversity of polypeptides in the proteome of any biological system changes rapidly and extensively in response to the state of the system. Building tools to measure proteome dynamics with single-molecule resolution is challenging because both single-molecule sensitivity and a high dynamic range are desired to comprehensively analyze the complex mixtures of polypeptides in a proteome. Moreover, single molecule measurements are prone to stochastic effects that are not apparent when measuring ensembles of molecules. Polypeptides being relatively large molecules display highly variable structural dynamics, the result of which can manifest as high stochasticity in detection of these molecules individually. This in turn can impact the quality of assay results. Proteome research is in need of an assay platform that is capable of measuring tens-of-billions of individual molecules in parallel with single-molecule sensitivity, as well as the accurate capture of both low-abundance and high-abundance polypeptides. The present disclosure addresses this need and provides other advantages as well. SUMMARY The present disclosure provides a method of identifying a polypeptide. The method can include steps of (a) attaching a polypeptide to a particle or solid support, thereby producing an immobilized polypeptide having a plurality of amino acids linked to the particle or solid support; (b) fragmenting the immobilized polypeptide, whereby the particle or solid support is attached to a set of fragments of the polypeptide; (c) performing a binding assay including contacting the set of fragments with a plurality of affinity reagents and detecting binding of affinity reagents of the plurality of affinity reagents to the set of fragments; and (d) identifying the polypeptide from results of the binding assay. Optionally, a method of identifying polypeptides can include steps of (a) attaching a polypeptide to a particle or solid support, thereby producing an immobilized polypeptide having a plurality of amino acids linked to the particle or solid support; (b) performing a binding assay including contacting the immobilized polypeptide with a plurality of affinity reagents and detecting binding of affinity reagents of the plurality of affinity reagents to the polypeptide; (c) fragmenting the immobilized polypeptide, whereby the particle or solid support is attached to a set of fragments of the polypeptide; (d) performing a second binding assay including contacting the set of fragments with a plurality of affinity reagents and detecting binding of affinity reagents of the plurality of affinity reagents to the set of fragments; and (e) identifying the polypeptide from results of the binding assay and second binding assay. Further optionally, a method of identifying a polypeptide can include steps of (a) attaching a polypeptide to a particle or solid support, wherein the polypeptide is from a biological sample, thereby producing an immobilized polypeptide having a plurality of amino acids linked to the particle or solid support; (b) fragmenting the immobilized polypeptide, whereby the particle or solid support is attached to a set of fragments of the polypeptide; (c) performing a binding assay including contacting the set of fragments with a plurality of affinity reagents and detecting binding of affinity reagents of the plurality of affinity reagents to the set of fragments; (d) identifying the polypeptide from results of the binding assay; (e) attaching a second polypeptide to a second particle or second solid support, wherein the second polypeptide is from the biological sample, thereby producing a second immobilized polypeptide comprising a plurality of amino acids linked to the second particle or second solid support; (f) fragmenting the second immobilized polypeptide, whereby the second particle is attached to a second set of fragments of the second polypeptide; and (g) performing a binding assay including contacting the se