Single-Cell Proteomic Assay Using Aptamer Barcoding
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Solution Overview
Problem
Current methods for single cell proteomics are limited in their ability to perform highly multiplexed analysis of proteins at high throughput, with existing techniques like flow cytometry and mass spectrometry facing challenges such as spectral overlap, limited isotope tags, and low throughput.
Innovation Solution
The development of oligonucleotide-adorned solid supports that use aptamers and Unique Molecular Identifiers (UMIs) for the identification of proteins expressed in single cells, allowing for high-throughput multiplex detection and sequencing-based readouts, enabling the analysis of protein-protein and protein-RNA interactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If flow cytometry is used for protein detection, then multiplex detection capability is improved, but spectral overlap limits the number of proteins that can be detected simultaneously
Solution Approach 1:
The patent introduces oligonucleotide tags as intermediary molecules that link proteins to sequencing-readable barcodes. Instead of directly detecting multiple proteins with fluorescent tags that suffer from spectral overlap, the invention uses oligonucleotide intermediaries that can be sequenced with high precision, thereby resolving the spectral overlap limitation while maintaining multiplex capability
Solution Approach 2:
The patent replaces the optical detection mechanism of flow cytometry with a sequencing-based detection system. By substituting fluorescent tag detection with oligonucleotide sequence analysis, the invention eliminates spectral overlap issues while enabling higher multiplexing capacity through the combinatorial power of sequence-based identification
2Adaptability or versatility
If mass cytometry is used for protein detection, then multiplex detection capability is improved, but limited isotope tags constrain the number of proteins that can be analyzed
Solution Approach 1:
The patent creates a universal oligonucleotide tag system that can identify any protein through sequencing, replacing the limited set of isotope tags. Each protein is tagged with a unique oligonucleotide sequence from a large combinatorial library, allowing thousands of proteins to be distinguished simultaneously without being constrained by the small number of available isotopic labels
Solution Approach 2:
The patent changes the identification parameter from isotopic mass (limited to ~20-50 distinct tags) to nucleotide sequence (enabling thousands to millions of distinct tags). By transforming the tagging system from mass-based to sequence-based identification, the invention dramatically expands the multiplexing capacity while maintaining compatibility with single-cell analysis
3Measurement precision
If mass spectrometry is used for proteome analysis, then quantitative analysis of entire proteomes is achieved, but large amounts of protein/cells are required and throughput is limited
Solution Approach 1:
The patent segments the proteome analysis process into two stages: (1) oligonucleotide-tagged proteins are captured and enriched in a preparatory step, and (2) pooled samples from many cells are sequenced simultaneously. This segmentation allows quantitative analysis to be achieved with minimal cells while dramatically increasing throughput through parallel processing of pooled samples
Solution Approach 2:
The patent performs preliminary enrichment and tagging of proteins with oligonucleotide labels before sequencing analysis. By pre-tagging proteins with sequencing-compatible oligonucleotides and performing preliminary purification steps, the invention enables quantitative proteome analysis with far fewer cells than traditional mass spectrometry, while the pooled sequencing approach subsequently achieves high throughput
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 simultaneous analysis of protein expression and gene expression in single cells with high multiplexity and throughput, providing detailed insights into cellular processes and interactions.
Implementation Method 1
an aptamer specific to the target barcode sequence configured for binding to a target protein
Implementation Method 2
The aptamers may further comprise a photoreactive group configured for covalently linking the aptamers to captured proteins upon experimentally defined illumination
Data Source
AI summary
The application relates to proteome analysis in single cells. Specifically, disclosed are high throughput methods of detecting proteins in single cells using barcoding, aptamers and single cell sequencing. Solid supports used in recording the cell-of-origin of target proteins and target proteins expressed in the cell-of-origin are disclosed. Additionally, methods of detecting proteins and mRNA in single cells are disclosed. Additionally, methods of detecting protein interactions are disclosed. Additionally, methods of detecting post translationally modified proteins in single cells are disclosed. The application also relates to solid supports or beads and methods of producing said solid supports or beads for use in the described methods.