Fluorescent Labeling for Trace Protein Detection
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Solution Overview
Problem
Current methods for detecting and identifying trace amounts of expressed proteins and peptides are labor-intensive, time-consuming, and suffer from low sensitivity due to the interference of non-target protein/peptide fragments, particularly in multi-dimensional HPLC and ICAT techniques.
Innovation Solution
A method involving fluorescent labeling of proteins/peptides, followed by selective separation and enzymatic hydrolysis, with mass spectrometry analysis and database collation for structural identification, allowing for high-sensitivity detection and separation of trace proteins/peptides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If multi-dimensional HPLC is used for separating and identifying protein/peptide, then separation capability is improved, but labor and time consumption increase considerably
Solution Approach 1:
The patent applies preliminary action by performing fluorescent labeling on target proteins/peptides before the separation process. This pre-marking allows for direct detection during HPLC separation, eliminating the need for post-separation identification steps and significantly reducing analysis time while maintaining separation capability
Solution Approach 2:
The patent utilizes fluorescent labeling to introduce optical properties to target proteins/peptides. The fluorescent tags enable detection through fluorescence signals during HPLC separation, allowing for real-time monitoring and identification without additional labor-intensive steps, thus improving analysis speed
2Adaptability or versatility
If ICAT method is used for comprehensive analysis of protein/peptide, then analysis comprehensiveness is improved, but fragments of non-target protein/peptide impair detection of target trace protein/peptide
Solution Approach 1:
The patent applies local quality by using fluorescent labeling that specifically targets certain functional groups (such as amino groups or thiol groups) on proteins/peptides. This selective labeling ensures that only proteins/peptides with the target functional groups are marked, allowing for specific detection without interference from non-target fragments, thus improving detection sensitivity while maintaining comprehensive analysis capability
Solution Approach 2:
The patent introduces fluorescent tags as intermediaries between the target proteins/peptides and the detection system. These fluorescent intermediaries provide specific optical signals that can be detected with high sensitivity, enabling the detection of trace target proteins/peptides even in the presence of大量 non-target fragments
3Reliability
If all thiol-containing protein/peptide are subjected to enzymatic hydrolysis, then complete digestion is achieved, but fragments of non-target protein/peptide present in large amounts impair detection and identification of target trace protein/peptide
Solution Approach 1:
The patent applies local quality by using fluorescent labeling that specifically targets certain functional groups (such as amino groups or thiol groups) on proteins/peptides. This selective labeling ensures that only proteins/peptides with the target functional groups are marked, allowing for specific detection without interference from non-target fragments, thus improving detection sensitivity while maintaining comprehensive analysis capability
Solution Approach 2:
The patent applies the extraction principle by isolating and detecting only the fluorescently labeled fractions during HPLC separation. This selective extraction of fluorescent signals from the complex digest mixture allows for the detection of trace target proteins/peptides without interference from abundant non-target fragments, thereby improving detection sensitivity while maintaining complete digestion
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 sensitive and efficient detection and identification of trace proteins/peptides, overcoming the limitations of previous methods by specifically extracting and analyzing fluorescently labeled fractions, thereby improving detection limits and analysis speed.
Implementation Method 1
a protein and/or peptide in a test sample is converted to a fluorescent derivative
Implementation Method 2
a protein and/or peptide in a test sample is converted to a fluorescent derivative
Implementation Method 3
the fluorescent derivative is separated by fluorescence detection
Implementation Method 4
the fluorescent fraction is applied to enzymatic hydrolysis
Implementation Method 5
the fluorescent fraction is applied to enzymatic hydrolysis
Implementation Method 6
the fractions are applied to mass spectrometry
Data Source
AI summary
A method of detection separation and identification for expressed trace protein/peptide; and a system therefor. There is provided a method of detecting, separating and identifying a minute amount of expressed protein and/or peptide, characterized in that a fluorescent derivative of protein and/or peptide contained in a test subject sample having been labeled with a fluorescence reagent is applied to HPLC; a fluorescent fraction is collected and subjected to enzymatic hydrolysis; mass-spectrometry of the resultant fluorescence-labeled fragments and non-fluorescence-labeled fragments is carried out; and the thus obtained ion molecular weight information on each of the fragments is collated with an available protein and/or peptide fragment database to thereby accomplish a structural analysis. Further, there is provided an identification system therefor.