BODIPY-based dyes for digital spatial proteomics
Inventors
BEELER, Aaron Beaty • Emili, Andrew • LENIHAN, Jason Michael • HENDRICK, Nathaniel Ryan • CHEN, Ichun Anderson
Assignees
Publication Number
US-12181479-B2
Publication Date
2024-12-31
Expiration Date
2042-12-22
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Abstract
The disclosure provides probes comprising dipyrromethane-BF2 derivatives which exhibits different fluorescent spectral properties when conjugated to the amino acids, compositions and kits comprising same. The disclosure also provides methods for detecting/identifying amino acids and sequencing polypeptide molecules by conjugating a dipyrromethane-BF2 derivative which exhibits different fluorescent spectral properties when conjugated to the amino acids.
Core Innovation
The invention provides probes comprising dipyrromethane-BF2 derivatives, including BODIPY dyes, that display different fluorescent spectral properties when conjugated to different amino acids. These probes and related kits allow for the detection, identification, and sequencing of amino acids and polypeptide molecules by measuring the specific spectral properties induced upon the probe–amino acid conjugation.
Current methods lack sufficient reagents, assays, and multiplex capabilities to efficiently detect, identify, and sequence amino acids within complex biological samples, or to perform imaging and quantification of multiple proteins simultaneously. The disclosed invention addresses these needs by presenting novel dyes and their use in methods capable of distinguishing and quantifying various amino acids or proteins in both solution and biological contexts.
The methods include conjugating the dipyrromethane-BF2-derived probe to an amino acid, detecting the resulting fluorescent properties, and identifying the amino acid by comparison to reference spectral profiles. Probes can be conjugated to free amino acids or to terminal amino acids within polypeptides, enabling peptide sequencing by iterative labeling and cleavage of terminal residues. The methods are extensible to multiplex detection, meaning multiple amino acids or polypeptides can be analyzed simultaneously.
Claims Coverage
The independent claims encompass two central inventive features directed at methods for identifying amino acids in polypeptides and for sequencing polypeptides using dipyrromethane-BF2 derivative probes.
Method for identifying amino acids in polypeptides using dipyrromethane-BF2 derivative probes
A method that includes: - Conjugating a probe comprising a dipyrromethane-BF2 derivative, which includes a labile or reactive group and exhibits different fluorescent spectral properties when conjugated to amino acids, to amino acids at the N- or C-terminal of polypeptides in a plurality of amino acids, where the plurality is composed of different amino acids. - Detecting one or more fluorescent spectral properties of the probe conjugated to the different amino acids. - Identifying the amino acids by comparing the measured fluorescent spectral properties of the conjugated probe to a plurality of reference fluorescent spectral properties, each reference being characteristic of the probe conjugated to a specific amino acid.
Method for sequencing a polypeptide using dipyrromethane-BF2 derivative probes
A method that includes: 1. Conjugating a probe comprising a dipyrromethane-BF2 derivative (with a labile or reactive group and differential spectral properties for each amino acid) to a terminal amino acid of a polypeptide. 2. Detecting one or more fluorescent spectral properties of the probe conjugated to that terminal amino acid. 3. Identifying the amino acid by comparing the detected property to a plurality of reference fluorescent spectra. 4. Cleaving the conjugated terminal amino acid from the polypeptide. 5. Repeating the steps for sequencing at least a portion of the polypeptide.
These inventive features define methods allowing multiplexed amino acid identification and polypeptide sequencing through fluorescent spectral analysis using dipyrromethane-BF2 derivative probes with discriminatory properties based on the conjugated amino acid.
Stated Advantages
The invention enables identification and quantification of amino acids and proteins in complex biological samples.
It provides reagents and methods for simultaneous multiplex detection of different amino acids or proteins.
The technology allows for sequencing of polypeptides.
Documented Applications
Detection and identification of amino acids in biological fluids, tissues, organs, or cells.
Sequencing of polypeptide molecules, including proteins, in situ or in complex biological samples.
Research applications involving digital spatial proteomics and characterizing protein networks in biomedical contexts.
Use in diagnostic and/or therapeutic kits as described.
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