Protein Screening via Nested Library and Mass Spectrometry
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current protein screening and display methods are limited by the need for a physical genotype-phenotype linkage, which restricts the selection pressures and introduces selection biases due to the large size of entities like phage or ribosome, and are laborious and inefficient for identifying proteins with specific biophysical or pharmacological criteria.
Innovation Solution
The NestLink method involves attaching detection tags to a protein library, allowing for the identification and quantification of proteins that fulfill defined criteria without a physical genotype-phenotype linkage, by using a nested library approach where polypeptide-encoding sequences are associated with unique detection tags, enabling redundant tagging and mass spectrometry-based analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If display methods (phage, ribosome, or yeast display) are used to analyze entire protein pools, then throughput is enormously increased without much labour, but selection bias is introduced and selection pressures are restricted due to the large size of the display particle (100-fold larger than the actual binding molecule)
Solution Approach 1:
The invention separates the protein of interest from the nucleic acid encoding it, allowing the protein to be analyzed independently without being part of a large display particle. This segmentation enables the protein to respond to a broader range of selection pressures while maintaining high throughput capability through pooled analysis.
Solution Approach 2:
The invention uses an intermediary system where proteins are tagged with unique molecular tags that can be detected without requiring a physical genotype-phenotype linkage. This intermediary tagging system allows for high-throughput analysis while avoiding the limitations of large display particles.
2Quantity of substance
If display methods are used with large entities like phage or ribosome, then entire protein pools can be processed, but selection bias is introduced due to the enormous size of the display particle
Solution Approach 1:
The invention extracts the protein from the context of large display particles and analyzes it independently. By taking out the protein from the phage or ribosome complex, the measurement reflects the actual protein properties without the confounding influence of the large display particle size.
3Measurement precision
If screens analyze proteins one by one, then individual binder candidates can be identified and characterized, but the process is very laborious and limited to a comparatively low number of tests
Solution Approach 1:
The invention combines the advantages of individual protein analysis with pooled processing. Multiple proteins are analyzed together in pools using unique molecular tags, maintaining the precision of individual characterization while achieving the throughput of pooled analysis.
Solution Approach 2:
The invention creates a universal tagging system that can be applied to any protein in the library, enabling all proteins to be analyzed simultaneously through a single detection method. This multi-functional approach allows individual protein characterization at scale.
Data Source
AI summary
The invention relates to a method for identifying and quantifying a polypeptide from a library of polypeptides. The method comprises the steps of: 1—providing a polypeptide library and a detection tag library, 2—generating a nested library comprising the polypeptides and the detection tags, 3—sequencing the nested library, 4—selecting a member of the nested library in one or several selection steps that are independent of a physical genotype-phenotype linkage, 5—isolating the detection tag from the selected polypeptide, 6—identifying and quantifying the detection tag by mass spectrometry, 7—obtaining the sequence of the selected polypeptide. The invention also relates to a collection of polypeptides, a collection of detection tags, and a collection of plasmid vectors.