Sequencing-Based Proteomics for High-Throughput Protein Validation
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Solution Overview
Problem
Current methods for validating protein expression and interactions are laborious and rely on high-quality antibodies, making it difficult to assess protein abundance, localization, modification, and interaction on a proteome-wide scale.
Innovation Solution
A sequencing-based proteomics method that combines deep sequencing with the CRISPR/Cas9 system to create a cell library where each cell expresses a fusion protein with a detectable marker, allowing for the quantification and localization of proteins on a proteome-wide scale.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard validation methods (western blot, co-immunoprecipitation, immunofluorescence microscopy) are used, then protein validation can be performed, but the process becomes laborious and requires high-quality antibodies
Solution Approach 1:
The patent replaces traditional mechanical and chemical validation methods (western blot, co-immunoprecipitation, immunofluorescence microscopy) with a sequencing-based detection system. By integrating detectable markers (fluorescent proteins, epitopes, enzymes) into target genes and using deep sequencing to detect these markers, the system substitutes antibody-based detection with sequence-based detection, eliminating the need for high-quality antibodies while maintaining validation accuracy and enabling high-throughput analysis
Solution Approach 2:
The patent creates a cellular copy system where target proteins are tagged with detectable markers that serve as detectable copies. Instead of using antibodies to detect native proteins, the system uses these marker copies (fluorescent proteins, epitopes, or enzymes) that can be detected through sequencing or readout mechanisms, thereby enabling high-throughput validation without the limitations of antibody availability
2Loss of information
If proteome-wide assessment is performed using traditional methods, then comprehensive protein information can be obtained, but the process requires extensive research and is not scalable
Solution Approach 1:
The patent creates a universal detection platform that can assess multiple protein parameters (abundance, localization, modification, interaction) simultaneously through a single sequencing-based approach. By using standardized detectable markers integrated into target genes, the system provides multi-functional assessment capabilities that replace multiple separate research processes, enabling comprehensive proteome-wide information获取 in a scalable, high-throughput manner without extensive time investment
3Measurement precision
If high-quality antibodies are used for protein detection, then detection accuracy is improved, but the method becomes limited to proteins with available antibodies
Solution Approach 1:
The patent replaces antibody-based detection with a sequencing-based detection system that uses detectable markers (fluorescent proteins, epitopes, enzymes) integrated into target genes. This substitution eliminates the dependency on antibody availability while maintaining detection accuracy through sequence-based identification and readout mechanisms, thereby expanding protein detection coverage to include all proteins for which genomic information is available
Solution Approach 2:
The patent implements a self-service detection system where the cellular system itself provides the detection capability through endogenously expressed detectable markers. Instead of requiring external antibodies, the cells express their own detectable markers (fluorescent proteins, epitopes, or enzymes) that are directly detectable through sequencing or readout mechanisms, enabling universal protein detection without external reagent dependency
Data Source
AI summary
The invention provides a cell library for use in detecting protein expression comprising a plurality of cells, wherein each cell comprises a polynucleotide sequence encoding a detectable marker integrated into the genome of the cell in frame with a protein coding gene selected from a set of target genes, wherein the library comprises more than one cell tagged at each target gene, as well as a cell library for use in detecting protein interactions between a protein of interest and a set of target proteins and a cell library for use in detecting protein modifications. The invention also provides methods of constructing a cell library for use in proteomics, as well as methods for sequencing integration sites of a donor sequence inserted into the genome of a cell. Also provided are systems for analysis of proteins in a cell and kits comprising vectors for tagging a population of cells and for performing proteomics studies.