Protein sequencing methods and reagents
AssigneeThe Governing Council of the University of Toronto
InventorAndrew Emili
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Abstract
Described are optical methods and reagents for sequencing polypeptides. A probe that exhibits different spectral properties when conjugated to different N-terminal amino acids is conjugated to the N-terminal amino acid of a polypeptide. Sequentially detecting one or more spectral properties of the probe conjugated to the N-terminal amino acid and cleaving the N-terminal amino acid produces sequence information of the polypeptide. The use of super-resolution microscopy allows for the massively parallel sequencing of individual polypeptide molecules in situ such as within a cell. Also described are probes comprising hydroxymethyl rhodamine green, an isothiocyanate group and a protecting group.
Claims10
A method of sequencing a polypeptide comprising:
- (a) conjugating a molecule to a terminal amino acid of the polypeptide, wherein the molecule exhibits different fluorescent spectral properties when conjugated to different terminal amino acids;
- (b) detecting one or more fluorescent spectral properties of the molecule conjugated to the terminal amino acid;
- (c) identifying a corresponding terminal amino acid of the polypeptide by comparing the one or more fluorescent spectral properties of the molecule conjugated to the terminal amino acid of the polypeptide to a plurality of reference fluorescent spectral properties, wherein each reference fluorescent spectral property of the plurality of reference fluorescent spectral properties is representative of the molecule conjugated to a different terminal amino acid;
- (d) cleaving the terminal amino acid of the polypeptide;
- and
- (e) sequentially repeating (a) to (d) to determine a sequence of at least a portion of the polypeptide, wherein the molecule in each subsequent iteration of (a) has an identical structure to the molecule in a first iteration of (a), and wherein the sequence of the at least the portion of the polypeptide comprises at least two different amino acids.
Dependencies are inferred from the claim text.