Polypeptide Identification via Sequence-Specific Hydrolysis and Affinity Enrichment
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Solution Overview
Problem
Current methods for proteomic analysis of complex biological samples are time-consuming, labor-intensive, and costly due to the need for complex equipment and techniques like tandem MS and MS2 procedures, which are also ineffective in distinguishing between abundant and less abundant proteins in mixed samples.
Innovation Solution
A method involving sequence-specific hydrolysis of polypeptides, followed by affinity chromatography and MS1 mass spectrometry, which allows for the rapid identification of distinct polypeptides and mutant peptides using accurate mass measurement without the need for tandem MS or MS2 procedures, utilizing immobilized binding partners and standard peptides for confirmation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If tandem MS and MS2 procedures are used for proteomic analysis, then measurement precision is improved, but device complexity and cost increase
Solution Approach 1:
The patent segments the complex proteomic analysis into distinct phases: (1) peptide enrichment using immobilized binding partners that specifically capture target peptides from complex biological samples, and (2) simplified MS1 analysis of the enriched peptides. This segmentation allows the use of less complex equipment while maintaining identification accuracy by focusing MS resources on pre-enriched target peptides rather than analyzing the entire complex mixture.
Solution Approach 2:
The patent applies preliminary action by performing peptide enrichment and purification before mass spectrometry analysis. The immobilized binding partners are used to pre-capture and concentrate target peptides from complex samples, and standard peptides are added for confirmation. This preliminary enrichment step simplifies the subsequent MS analysis, allowing accurate identification using less complex MS1 procedures rather than requiring tandem MS or MS2.
2Manufacturing precision
If complex fractionation methods like 2-D gel electrophoresis are used, then separation precision is improved, but productivity decreases
Solution Approach 1:
The patent replaces complex mechanical separation systems (2-D gel electrophoresis, HPLC) with a biochemical enrichment approach using immobilized binding partners. Instead of physically separating all proteins in a complex mixture through multiple electrophoresis or chromatography steps, the method uses specific molecular recognition to enrich target peptides directly from the complex sample, followed by simplified MS1 analysis. This substitution dramatically reduces processing time while maintaining the ability to identify specific polypeptides and mutant peptides.
3Illumination intensity
If abundant proteins are present in biological samples, then signal intensity is improved, but detection of less abundant proteins deteriorates due to signal suppression
Solution Approach 1:
The patent introduces immobilized binding partners as intermediaries that specifically recognize and capture target peptides from complex biological samples. These binding partners act as mediators that selectively enrich low-abundance target peptides while leaving abundant proteins in the supernatant. The enriched peptides are then analyzed by MS1, allowing detection of low-abundance polypeptides without signal suppression from abundant proteins. Standard peptides are also used as intermediaries for confirmation of identification.
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
This approach enables rapid and cost-effective identification of polypeptides and mutant peptides in complex biological samples, reducing the complexity of the analysis and eliminating the requirement for specialized and expensive equipment, while providing accurate mass-based identification.
Implementation Method 1
contacting the biological sample with a hydrolyzing agent, wherein the hydrolyzing agent is capable of hydrolyzing the distinct polypeptide in a sequence-specific manner
Implementation Method 2
bringing the hydrolyzed sample in contact with a substrate comprising at least one immobilized binding partner, wherein the at least one immobilized binding partner is capable of specifically binding the distinct peptide
Implementation Method 3
Mass spectrometry (MS) is an important method for the characterization of proteins in biological samples. MS involves ionizing chemical compounds to generate charged molecules or molecule fragments and measurement of their mass or mass-to-charge ratios
Data Source
AI summary
The invention relates to a method for determining the presence of at least one distinct polypeptide in a biological sample comprising contacting the biological sample with a hydrolyzing agent, wherein the hydrolyzing agent is capable of hydrolyzing the distinct polypeptide in a sequence-specific manner such that at least one distinct peptide having a predetermined peptide measured accurate mass would result if the at least one distinct polypeptide were present in the biological sample, to obtain a hydrolyzed sample; bringing the hydrolyzed sample in contact with a substrate comprising at least one immobilized binding partner, wherein the at least one immobilized binding partner is capable of specifically binding the distinct peptide; removing the hydrolyzed sample from the substrate in a manner such that the distinct peptide would remain bound to the immobilized binding partner; contacting the substrate with an elution solution, wherein the distinct peptide would dissociate from the immobilized binding partner into the elution solution; subjecting a portion of the elution solution to liquid chromatography to segregate a plurality of molecules in the portion of the elution solution to obtain sorted molecules; determining the measured accurate mass of at least one sorted molecule present in the elution solution; and determining the presence of the at least one distinct polypeptide in the biological sample when a measured accurate mass of at least one molecule is substantially equal to the predetermined peptide measured accurate mass.