GB2610078A - Single molecule N-terminal sequencing using electrical signals - Google Patents

Single molecule N-terminal sequencing using electrical signals Download PDF

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Publication number
GB2610078A
GB2610078A GB2215304.3A GB202215304A GB2610078A GB 2610078 A GB2610078 A GB 2610078A GB 202215304 A GB202215304 A GB 202215304A GB 2610078 A GB2610078 A GB 2610078A
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United Kingdom
Prior art keywords
polypeptide
amino acid
array
field effect
effect transistor
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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GB2215304.3A
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GB202215304D0 (en
Inventor
Somekh Tal
Booth Simpson Zachary
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Erisyon Inc
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Erisyon Inc
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Publication of GB202215304D0 publication Critical patent/GB202215304D0/en
Publication of GB2610078A publication Critical patent/GB2610078A/en
Pending legal-status Critical Current

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    • 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
    • G01N33/6818—Sequencing of polypeptides
    • G01N33/6824—Sequencing of polypeptides involving N-terminal degradation, e.g. Edman degradation
    • 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
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Nanotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Peptides Or Proteins (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present disclosure provides methods and systems for polypeptide (e.g., protein) sequencing or identification using field effect transistor (FET) arrays.

Claims (57)

1. A method for polypeptide sequencing, comprising: a) providing an array having a polypeptide immobilized thereto, wherein said polypeptide is adjacent to a sensor; b) subjecting said polypeptide to conditions sufficient to remove an amino acid from said polypeptide in a solution; c) using said sensor to measure a charge, conductivity, or impedance, or change thereof, in said solution subsequent to removal of said amino acid from said polypeptide; and d) using at least said charge, conductivity, or impedance, or change thereof, to identify a sequence of said polypeptide.
2. The method of claim 1, wherein (b) comprises subjecting said polypeptide to Edman degradation.
3. The method of claim 1, wherein (b) comprises mixing said polypeptide with a diactivated phosphate or phosphonate to form a reaction mixture, and mixing said reaction mixture with an acid to remove said amino acid.
4. The method of claim 3, wherein said diactivated phosphate or phosphonate is a dihalophosphate ester.
5. The method of claim 1, further comprising repeating (b) and (c) to measure an additional charge, conductivity, or impedance, or change thereof, in said solution subsequent to removal of an additional amino acid from said polypeptide.
6. The method of claim 1, wherein (d) comprises identifying said amino acid removed in (b).
7. The method of claim 1, wherein (d) comprises identifying a chemical modification of said amino acid removed in (b).
8. The method of claim 7, wherein said chemical modification comprises a post-translational modification.
9. The method of claim 7, wherein said chemical modification comprises a chemical label.
10. The method of claim 7, wherein said chemical modification comprises a disulfide.
11. The method of claim 1, wherein (a) comprising immobilizing another polypeptide to said array, thereby providing said another polypeptide to said array.
12. The method of claim 11, wherein said another polypeptide is derived from a plurality of polypeptides or a protein.
13. The method of claim 12, wherein said plurality of polypeptides are provided to said array in a Poisson distribution.
14. The method of claim 12, wherein said plurality of polypeptides are provided to said array in a super-Poisson distribution.
15. The method of claim 1, wherein said measuring comprises at most 5 seconds of signal averaging.
16. The method of claim 1, wherein said change in said impedance is at least 1 kQ.
17. The method of claim 1, wherein said change in said conductivity is at least 106 Scm 1.
18. The method of claim 1, wherein said sensor comprises a field effect transistor (FET).
19. The method of claim 18, wherein said FET is selected from a group consisting of ion- sensitive field effect transistor (ISFET), metal-oxide-semiconductor field effect transistor (MOSFET), enzyme field effect transistor (EnFET), chemically-sensitive field effect transistor (ChemFET), a carbon nanotube field effect transistor (CNFET), immuno-field effect transistor (ImmunoFET), or a biologically sensitive field effect transistor (BioFET).
20. The method of claim 19, wherein said FET comprises a floating gate.
21. The method of claim 20, wherein said floating gate has a size greater than 1 nm2 having a trapped charge of less than 240 V.
22. The method of claim 19, wherein said FET occupies an area of up to 1 mm2.
23. The method of claim 1, wherein said sensor measures said charge or change thereof.
24. The method of claim 1, wherein said sensor measures said conductivity or change thereof.
25. The method of claim 1, wherein said sensor measures said impedance or change thereof.
26. The method of claim 1, wherein said array comprises a support, and wherein said polypeptide is immobilized to said support.
27. The method of claim 26, wherein said support comprises a bead.
28. The method of claim 26, wherein said support comprises a surface of a well.
29. The method of claim 28, wherein said well is among a plurality of wells.
30. The method of claim 29, wherein said plurality of wells comprises at least two wells.
31. The method of claim 29, wherein said plurality of wells comprises at least 10,000 wells.
32. The method of claim 1, wherein said polypeptide is coupled to a capture moiety coupled to said array.
33. The method of claim 32, wherein said array comprises a plurality of individually addressable sites, and wherein said polypeptide is immobilized to an individually addressable site of said plurality of individually addressable sites.
34. The method of claim 33, wherein said polypeptide is covalently coupled to said array.
35. The method of claim 33, wherein said polypeptide is ionically coupled to said array.
36. The method of claim 1, wherein said sensor comprises a carbon nanotube transistor.
37. The method of claim 1, wherein said array comprises a plurality of sites, and wherein in (a) said polypeptide is immobilized to a single site of said plurality of sites.
38. The method of claim 1, wherein (b) and (c) are performed in substantially real time.
39. The method of claim 1, wherein (d) further comprises identifying a conformation or a chemical modification of said polypeptide.
40. The method of claim 1, wherein (d) further comprises identifying a disulfide bond of said polypeptide.
41. The method of claim 1, wherein said polypeptide is coupled to an engineered side chain coupled to said array.
42. The method of claim 41, wherein said engineered side chain comprises a covalent bond between a post translational modification on an amino acid residue of said polypeptide and a labeling reagent.
43. The method of claim 42, wherein said post translational modification comprises phosphorylation, glycosylation, nitrosylation, citrullination, sulfenylation, or trimethylation.
44. The method of claim 41, wherein said engineered side chain comprises an ionic bond between said post translational modification on said amino acid residue of said peptide or protein and a labeling reagent.
45. The method of claim 44, wherein said post translational modification on said amino acid residue comprises phosphorylation, glycosylation, nitrosylation, citrullination, sulfenylation, or trimethylation.
46. The method of claim 41, wherein said engineered side chain is spectrally activated and a spectral signature of said attached labelled peptide is recorded.
47. The method of claim 46, wherein said spectrally activated side chain is stimulated by a field effect transistor.
48. The method of claim 47, wherein said stimulation is pulsed.
49. The method of claim 47, wherein said stimulation is constant.
50. The method of claim 46, wherein said spectrally activated side chain is stimulated by an ion-sensitive field effect transistor.
51. The method of claim 46, wherein said spectrally activated side chain identifies a unique spectral signature upon stimulation.
52. The method of claim 46, wherein said unique spectral signature that occurs from each recording device is analyzed to determine its origin.
53. The method of claim 52, wherein said spectral analysis is used to estimate concentrations of individual species of peptide present in the original mixture.
54. The method of claim 41, wherein the entire collection of polypeptides loses one amino acid from said polypeptide to detect an additional signal indicative of a change in spectra subsequent to removal of said additional amino acid from said polypeptide.
55. The method of claim 54, wherein said unique spectral signature(s) over all addressable units are collected between peptide degradation cycles.
56. A method for polypeptide sequencing, comprising: (a) providing an array having a polypeptide immobilized thereto, wherein said polypeptide is adjacent to a sensor; (b) subjecting said polypeptide to conditions sufficient to remove an amino acid from said polypeptide; (c) using said sensor to measure a non-optical signal in said solution subsequent to removal of said amino acid from said polypeptide; and (d) using at least said non-optical signal to identify a sequence of said polypeptide.
57. A method for determining a sequence of an unlabeled peptide, said method comprising: a) removing an amino acid from said unlabeled peptide; b) identifying a change in an electrical signal of a FET disposed adjacent to said peptide; c) repeating a) and b) at least once, thereby determining said sequence of said unlabeled peptide.
GB2215304.3A 2020-04-13 2021-04-13 Single molecule N-terminal sequencing using electrical signals Pending GB2610078A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202063009240P 2020-04-13 2020-04-13
PCT/US2021/027155 WO2021211631A2 (en) 2020-04-13 2021-04-13 Single molecule n-terminal sequencing using electrical signals

Publications (2)

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GB202215304D0 GB202215304D0 (en) 2022-11-30
GB2610078A true GB2610078A (en) 2023-02-22

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WO (1) WO2021211631A2 (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
LU501669B1 (en) 2022-03-15 2023-09-20 Luxembourg Inst Science & Tech List Identification of amino acids or short peptides.
LU501668B1 (en) 2022-03-15 2023-09-22 Luxembourg Inst Science & Tech List Method and device for cleaving and/or sequencing a peptide
EP4394371A1 (en) * 2022-12-30 2024-07-03 Imec VZW A bio-fet-based peptide sequencing method and device

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009158006A2 (en) * 2008-06-26 2009-12-30 Ion Torrent Systems Incorporated Methods and apparatus for detecting molecular interactions using fet arrays
US20140273004A1 (en) * 2013-03-15 2014-09-18 Washington University Molecules and methods for iterative polypeptide analysis and processing
WO2019063829A1 (en) * 2017-09-29 2019-04-04 Melief Jeroen Immunogenic composition for the treatment of cancer
WO2019178033A1 (en) * 2018-03-12 2019-09-19 Arbor Biotechnologies, Inc. Ultrahigh throughput protein discovery

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB201715684D0 (en) * 2017-09-28 2017-11-15 Univ Gent Means and methods for single molecule peptide sequencing

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009158006A2 (en) * 2008-06-26 2009-12-30 Ion Torrent Systems Incorporated Methods and apparatus for detecting molecular interactions using fet arrays
US20140273004A1 (en) * 2013-03-15 2014-09-18 Washington University Molecules and methods for iterative polypeptide analysis and processing
WO2019063829A1 (en) * 2017-09-29 2019-04-04 Melief Jeroen Immunogenic composition for the treatment of cancer
WO2019178033A1 (en) * 2018-03-12 2019-09-19 Arbor Biotechnologies, Inc. Ultrahigh throughput protein discovery

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MAITI et al. Bifunctional aryloxyphosphoramidate prodrugs of 2'-C-Me-Uridine: synthesus and anti-HCV activity. Organic & Biomolecular Chemistry. 21 September 2016, Vol, 14, No. 37, pg 8743-8757; especially pg 3, col 2, para 3 *

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WO2021211631A3 (en) 2021-12-02
WO2021211631A2 (en) 2021-10-21

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