Single-Molecule Peptide Sequencing With Fluorescent Labels
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Solution Overview
Problem
Current methods for sequencing peptides, particularly Edman degradation, are limited by the length of peptides that can be sequenced and cannot identify individual peptides within a mixture, especially if the N-terminal amino acid is modified or buried, and lack high-throughput methods for protein identification and quantitation in complex samples.
Innovation Solution
A method involving selective labeling of amino acids on immobilized peptides with specific fluorophores, followed by Edman degradation and single-molecule detection, allowing for the sequencing and identification of intact peptides in a mixture, including those with unnatural amino acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Edman degradation is used for peptide sequencing, then the N-terminal amino acid can be identified, but the method fails when the N-terminal amino acid is modified or buried
Solution Approach 1:
The invention extracts the N-terminal amino acid from the peptide chain using selective cleavage chemistry (e.g., Edman degradation or specific proteases) before identification. This allows the N-terminal residue to be analyzed separately, overcoming the limitation of direct sequencing when the N-terminus is modified or buried within the protein structure.
Solution Approach 2:
The invention introduces intermediary reagents or tags that bind to the N-terminal amino acid, enabling its identification even when modified. These intermediaries act as mediators between the modified N-terminal residue and the detection system, allowing indirect identification of the N-terminal amino acid type.
2Measurement precision
If traditional peptide sequencing methods are used, then individual peptides can be analyzed, but high-throughput sequencing and quantitation in complex mixtures cannot be achieved
Solution Approach 1:
The invention segments the complex peptide mixture into individual peptide components through immobilization on solid supports or beads. Each peptide is then processed independently through sequencing and identification steps, enabling parallel high-throughput analysis while maintaining individual peptide resolution. This segmentation allows millions of peptides to be analyzed simultaneously.
Solution Approach 2:
The invention creates copies of peptide information through mass spectrometry signaling and detection systems. Each peptide's sequence information is converted into detectable signals (e.g., mass spectra, fluorescent signals) that can be amplified and detected, enabling high-throughput quantitation and identification without requiring direct manual analysis of each peptide.
3Measurement precision
If labeling methods are used to identify amino acids, then fluorescence detection can be achieved, but the complexity of the labeling process increases
Solution Approach 1:
The invention employs universal labeling reagents that can tag multiple types of amino acids with a single reagent system. For example, amino acid-specific reagents can label different amino acid types with distinct fluorescent labels in a standardized protocol, reducing the need for separate complex labeling procedures for each amino acid type while maintaining high identification sensitivity.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables rapid, high-throughput sequencing and quantitation of individual peptides and proteins in complex samples, capable of identifying a majority of proteins from species like yeast and human proteomes, even with buried or modified N-terminal amino acids.
Implementation Method 1
labeling the N-terminal amino acid with a first label and labeling an internal amino acid with a second label... In some embodiments, the labels are fluorescent labels
Data Source
AI summary
Methods of identifying a sequence of a polypeptide within a heterogenous mixture of polypeptides, the polypeptide being immobilized to a support and having at least one labeled amino acid residue. Methods involve detecting at least one signal or signal change from the immobilized polypeptide and subjecting the polypeptide to conditions sufficient to remove at least one amino acid residue from the polypeptide.