N-terminal Amino Acid Sequencing via Spatial Segmentation
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Solution Overview
Problem
Current protein sequencing methods are low-throughput, computationally demanding, and require large sample amounts, limiting the ability to exhaustively sequence complex protein mixtures, especially in biological samples like blood or tissue extracts, due to ion-ion interference and biased detection.
Innovation Solution
The method involves affixing polypeptides to a substrate and contacting them with probes that selectively bind to N-terminal amino acid residues, allowing for the sequential detection and cleavage of these residues to determine the protein sequence, enabling both qualitative and quantitative analysis of protein expression in samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If mass spectrometry is used for protein sequencing, then speed and accuracy are improved, but sample amount requirement increases and throughput decreases
Solution Approach 1:
The invention segments the protein sequencing process into individual single-molecule events occurring at spatially separated locations on a substrate. Each polypeptide molecule is immobilized at a distinct location and sequenced independently, allowing parallel processing of multiple molecules simultaneously. This segmentation enables high-throughput sequencing while maintaining single-molecule sensitivity and accuracy.
Solution Approach 2:
The invention transitions from temporal sequencing (one molecule at a time) to spatial parallelism (multiple molecules simultaneously at different locations). By arranging polypeptide molecules at spatially resolved positions on a substrate and using position-dependent detection, the system achieves massively parallel sequencing without sacrificing measurement precision.
2Measurement precision
If mass spectrometry is used for protein sequencing, then detection sensitivity is improved, but sample amount requirement increases
Solution Approach 1:
The invention isolates and sequences individual single polypeptide molecules at spatially separated locations on a substrate. By focusing detection resources on single molecules rather than bulk populations, the system achieves ultra-sensitive detection without requiring large sample amounts. Each molecular event is detected independently with high signal-to-noise ratio.
Solution Approach 2:
The invention creates spatial copies of the detection process across multiple substrate locations. Each location serves as an independent detection station for a single polypeptide molecule. This parallel copying of the detection function enables simultaneous analysis of many molecules with the sensitivity of single-molecule detection.
3Device complexity
If Edman degradation is used for protein sequencing, then equipment cost is reduced, but throughput and speed decrease
Solution Approach 1:
The invention replaces the sequential mechanical/chemical Edman degradation process with an optical detection system that reads amino acid sequences through fluorescently labeled probes. This substitution eliminates the need for sequential chemical cleavage steps, enabling parallel processing of multiple molecules simultaneously and dramatically increasing sequencing throughput while maintaining simplicity.
Solution Approach 2:
The invention transitions from sequential temporal processing (one amino acid at a time in Edman degradation) to parallel spatial processing (multiple amino acids detected simultaneously at different locations). By using position-dependent optical detection, the system achieves high-speed sequencing without complex equipment.
4Measurement precision
If sequential detection of N-terminal amino acids is used, then sequencing accuracy is improved, but time consumption increases
Solution Approach 1:
The invention maps the sequential detection process onto spatial dimensions. Multiple sequential detection events occur simultaneously at different spatial locations on the substrate. Each location performs sequential amino acid detection with high accuracy, while the overall system achieves parallel throughput by processing many sequences concurrently across the substrate array.
Solution Approach 2:
The invention performs preliminary immobilization of polypeptide molecules at spatially resolved locations on the substrate before detection begins. This preliminary positioning enables subsequent parallel processing without cross-contamination or sample loss, allowing sequential detection at each location to proceed efficiently without time-consuming sample handling between measurements.
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 the sequencing of single polypeptide molecules or multiple molecules simultaneously, facilitating the analysis of complex protein mixtures with improved sensitivity and accuracy, suitable for biological and environmental samples.
Implementation Method 1
contacting them with probes that selectively bind to N-terminal amino acid residues
Data Source
AI summary
The invention describes methods and reagents useful for sequencing polypeptide molecules. The method comprises affixing a polypeptide to a substrate and contacting the polypeptide with a plurality of probes that selectively bind to a terminal amino acid or a terminal amino acid derivative. Probes bound to the polypeptide molecule are then identified before cleaving the terminal amino acid or terminal amino acid derivative of the polypeptide. Also provided are probe compositions comprising (a) a ClpS variant comprising at least 80% sequence identity to SEQ ID NO: 1, wherein the ClpS variant binds to an N-terminal amino acid or a N-terminal amino acid derivative with a different selectivity than a wild-type ClpS; and (b) a detectable label.