# US 12,000,835 B2 · Methods and compositions for protein sequencing

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- Number: US 12,000,835 B2
- Document ID: US12000835B2
- Status: Granted
- Kind: B2
- Application number: 16/709,052
- Filed: 2019-12-10
- Published: 2020-07-02
- Granted: 2024-06-04
- Earliest claimed: 2018-11-15
- Assignees: Quantum-Si Incorporated
- Applicants: Quantum-Si Incorporated
- Inventors: Brian Reed; Jeremy Lackey; Haidong Huang
- Web page: https://patentgrep.com/patent/US12000835B2

## CPC

- G01N 33/581: {with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)}
- C07K 14/47: from mammals
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- G01N 1/28: Preparing specimens for investigation {including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q}(mounting specimens on microscopic slides G02B21/34; means for supporting the objects or the materials to be analysed in electron microscopes H01J37/20 {; laboratory gas handling apparatus B01L5/00})
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 33/58: involving labelled substances (G01N33/53 takes precedence)
- G01N 33/582: {with fluorescent label}
- G01N 33/6821: {involving C-terminal degradation}
- G01N 33/6824: {involving N-terminal degradation, e.g. Edman degradation}
- G16B 25/10: Gene or protein expression profiling; Expression-ratio estimation or normalisation
- G16B 40/00: ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- G16B 40/10: Signal processing, e.g. from mass spectrometry [MS] or from PCR
- G16B 50/30: Data warehousing; Computing architectures
- G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks}
- G01N 2458/00: Labels used in chemical analysis of biological material
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B 50/00: ICT programming tools or database systems specially adapted for bioinformatics
- 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)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

Aspects of the application provide methods of identifying and sequencing proteins, polypeptides, and amino acids, and compositions useful for the same. In some aspects, the application provides methods of obtaining data during a degradation process of a polypeptide, and outputting a sequence representative of the polypeptide. In some aspects, the application provides amino acid recognition molecules having a shielding element that enhances photostability in polypeptide sequencing reactions.

## Claims

### Claim 1

A method of identifying an amino acid of a polypeptide, the method comprising:
contacting a single polypeptide molecule with one or more amino acid recognition molecules that bind to the single polypeptide molecule, wherein at least one amino acid recognition molecule comprises an N-end rule pathway protein;
detecting a series of signal pulses indicative of association of the one or more amino acid recognition molecules with the single polypeptide molecule under polypeptide degradation conditions in a reaction mixture comprising a first aminopeptidase and a second aminopeptidase,
wherein the first aminopeptidase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 48, and the second aminopeptidase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 59 or 69; and
identifying a first type of amino acid in the single polypeptide molecule based on a first characteristic pattern in the series of signal pulses.

### Claim 2 (depends on 1)

The method of claim 1, wherein the first characteristic pattern comprises at least a portion of the series of signal pulses.

### Claim 3 (depends on 2)

The method of claim 2, wherein a signal pulse of the first characteristic pattern corresponds to an individual association event between an amino acid recognition molecule and the first type of amino acid.

### Claim 4 (depends on 3)

The method of claim 3, wherein the signal pulse of the first characteristic pattern comprises a pulse duration that is characteristic of a dissociation rate of binding between the amino acid recognition molecule and the first type of amino acid.

### Claim 5 (depends on 4)

The method of claim 4, wherein each signal pulse of the first characteristic pattern is separated from another by an interpulse duration that is characteristic of an association rate of amino acid recognition molecule binding.

### Claim 6 (depends on 1)

The method of claim 1, wherein the first characteristic pattern corresponds to a series of reversible amino acid recognition molecule binding interactions with the first type of amino acid in the single polypeptide molecule.

### Claim 7 (depends on 6)

The method of claim 6, wherein the series of reversible amino acid recognition molecule binding interactions comprises a reversible formation of one binary complex species at a terminus of the single polypeptide molecule.

### Claim 8 (depends on 1)

The method of claim 1, wherein the single polypeptide molecule comprises the first type of amino acid at a terminal position of the single polypeptide molecule.

### Claim 9 (depends on 8)

The method of claim 8, wherein the first characteristic pattern is indicative of the first type of amino acid at the terminal position and an amino acid type at a contiguous position.

### Claim 10 (depends on 1)

The method of claim 1, wherein each of the one or more amino acid recognition molecules comprises a detectable label.

### Claim 11 (depends on 10)

The method of claim 10, wherein the detectable label is a luminescent label or a conductivity label.

### Claim 12 (depends on 1)

The method of claim 1, wherein the single polypeptide molecule is immobilized to a surface.

### Claim 13 (depends on 1)

The method of claim 1, further comprising observing degradation of the single polypeptide molecule by the first or second aminopeptidase in the reaction mixture.

### Claim 14 (depends on 13)

The method of claim 13, further comprising identifying a second type of amino acid in the single polypeptide molecule based on a second characteristic pattern in the series of signal pulses.

### Claim 15 (depends on 14)

The method of claim 14, wherein the second characteristic pattern of signal pulses is indicative of removal of the first type of amino acid from the single polypeptide molecule.

### Claim 16 (depends on 1)

The method of claim 1, wherein the series of signal pulses is a series of real-time signal pulses.

### Claim 17 (depends on 1)

A system comprising:
at least one hardware processor; and
at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by the at least one hardware processor, cause the at least one hardware processor to perform the method of claim 1.

### Claim 18 (depends on 1)

At least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by at least one hardware processor, cause the at least one hardware processor to perform the method of claim 1.

### Claim 19 (depends on 1)

The method of claim 1, wherein signal pulses of the first characteristic pattern comprise a mean pulse duration of between about 1 millisecond and about 10 seconds.

### Claim 20 (depends on 19)

The method of claim 19, wherein the mean pulse duration is between about 10 milliseconds and about 100 milliseconds or between about 100 milliseconds and about 500 milliseconds.

### Claim 21 (depends on 1)

The method of claim 1, wherein the characteristic pattern of one type of amino acid is different from the characteristic pattern of another type of amino acid by a mean pulse duration of at least 10 milliseconds.

### Claim 22 (depends on 21)

The method of claim 21, wherein the characteristic pattern of one type of amino acid is different from the characteristic pattern of another type of amino acid by a mean pulse duration of between about 10 milliseconds and about 100 milliseconds or between about 100 milliseconds and about 10 seconds.

### Claim 23 (depends on 1)

The method of claim 1, wherein the reaction mixture comprises an amino acid recognition molecule and an aminopeptidase in a ratio of between about 10:1 and about 200:1.

### Claim 24 (depends on 1)

The method of claim 1, wherein the reaction mixture comprises an amino acid recognition molecule and an aminopeptidase in a ratio of between about 50:1 and about 150:1.

### Claim 25 (depends on 1)

The method of claim 1, wherein the N-end rule pathway protein is a Gid protein, a UBR box protein or UBR box domain-containing fragment thereof, a p62 protein or ZZ domain-containing fragment thereof, a ClpS protein, or a mutant or variant thereof.

### Claim 26 (depends on 1)

The method of claim 1, wherein the N-end rule pathway protein is a ClpS protein having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1.

### Claim 27 (depends on 1)

The method of claim 1, wherein the second aminopeptidase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 59.

### Claim 28 (depends on 1)

The method of claim 1, wherein the second aminopeptidase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 69.

### Claim 29 (depends on 1)

The method of claim 1, wherein the first aminopeptidase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 48, and the second aminopeptidase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 59 or 69.