Single Molecule Peptide Sequencing via Fluorescent Labeling
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Solution Overview
Problem
Current methods for large-scale identification and quantitation of specific proteins in complex mixtures are limited, particularly in sequencing intact peptides within a mixture of diverse peptides at the single molecule level, as existing techniques like Edman degradation are restricted to peptides with fewer than 50-60 amino acids and cannot sequence peptides in a mixture or with chemically modified N-terminal amino acids.
Innovation Solution
A method involving selective labeling of amino acids on immobilized peptides followed by successive cycles of labeling and removal of N-terminal amino acids, using fluorescent labels to produce unique patterns for identifying and quantitating individual peptides within a complex sample, capable of sequencing peptides up to 20 cycles to cover a significant fraction of the human and yeast proteomes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Edman degradation is used for peptide sequencing, then N-terminal amino acid identification is achieved, but peptide length is limited to fewer than 50-60 amino acids
Solution Approach 1:
The patent divides the peptide into multiple segments by performing sequential Edman degradation cycles, where each cycle removes and identifies one N-terminal amino acid. This segmentation approach allows the peptide to be analyzed in manageable increments, enabling sequencing of longer peptides that would otherwise exceed the 50-60 amino acid limitation of traditional single-cycle methods.
Solution Approach 2:
The patent employs periodic action through repeated cycles of Edman degradation, where the labeling and removal process is performed multiple times in succession. Each cycle provides sequence information for one amino acid position, and by repeating this periodic process, the method accumulates sequence data across the entire length of the peptide, overcoming the traditional length restriction.
2Measurement precision
If traditional sequencing methods are used, then peptide identification is achieved, but peptides in complex mixtures cannot be sequenced at the single molecule level
Solution Approach 1:
The patent uses fluorescent labeling to create detectable copies or markers on each peptide molecule. By attaching fluorescent labels to specific amino acid residues, the method creates optical copies that can be detected and tracked individually, allowing single-molecule sequencing even in complex mixtures where peptides cannot be physically separated.
Solution Approach 2:
The patent employs color changes through fluorescent labeling, where different fluorescent dyes are attached to different amino acid residues. This allows individual peptide molecules to be visually distinguished and tracked based on their fluorescent signals, enabling sequencing in complex mixtures by detecting the presence and position of specific colored markers on each molecule.
3Measurement precision
If high sensitivity detection is implemented, then single molecule level identification is achieved, but existing methods cannot handle peptides with chemically modified N-terminal amino acids
Solution Approach 1:
The patent applies parameter changes by modifying the chemical state of the peptide through fluorescent labeling. By changing the chemical parameters of specific amino acid residues (adding fluorescent groups), the method makes otherwise undetectable or unsequenceable modified peptides amenable to analysis, as the labeling creates detectable signals regardless of the original N-terminal modifications.
Solution Approach 2:
The patent uses fluorescent labels as intermediaries that mediate between the chemically modified peptide and the detection system. These intermediary labels attach to specific residues on the modified peptide, serving as detectable proxies that allow the sequencing process to proceed even when the N-terminal amino acid itself is chemically modified and would otherwise prevent standard detection.
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 the identification and quantitation of individual peptides in complex mixtures with high sensitivity, overcoming limitations of existing methods by allowing sequencing of longer peptides and those with chemically modified N-termini, and providing patterns that uniquely identify proteins within known proteomes.
Implementation Method 1
using fluorescent labels to produce unique patterns for identifying and quantitating individual peptides within a complex sample
Implementation Method 2
In the Edman procedure phenylisothiocyanate reacts quantitatively with the free amino group of a peptide to yield the corresponding phenylthiocarbamoyl peptide
Implementation Method 3
On treatment with anhydrous acid the N-terminal residue is split off as a phenylthiocarbamoyl amino acid, leaving the rest of the peptide chain intact
Data Source
AI summary
The present invention relates to methods for identifying amino acids in peptides. In one embodiment, the present invention contemplates 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. In other embodiments, the internal amino acid is lysine. In other embodiments, amino acids in peptides are identified based on the fluorescent signature for each peptide at the single molecule level.