Single-Cell Proteomics Using Oligonucleotide-Tagged Antibodies
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Solution Overview
Problem
Current methods for proteomic detection in single cells and cell populations are expensive, biased towards abundant proteins, not perfectly quantitative, and limited in multiplexed protein measurements, with existing antibody detection methods like immunofluorescence-based flow cytometry unable to resolve more than forty antibodies in a mixture.
Innovation Solution
A method involving contacting cells with multiple antibodies conjugated to oligonucleotides, separating single cells into compartments, amplifying unique molecular identifier sequences, and analyzing them to identify expression profiles, while removing duplicates, and optionally sequencing to determine protein expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If immunofluorescence-based flow cytometry is used for antibody detection, then the method is more quantitative and requires less input material, but it cannot resolve more than forty antibodies in a mixture due to fluorescence spectral signature limitations
Solution Approach 1:
The patent uses oligonucleotide tags as intermediaries between antibodies and detection systems. Each antibody is conjugated to a unique oligonucleotide tag that can be specifically amplified and detected by sequencing, allowing hundreds of antibodies to be resolved simultaneously without spectral overlap issues that limit flow cytometry to forty antibodies.
Solution Approach 2:
The patent replaces the optical detection system (fluorescence spectroscopy) with a molecular biology-based detection system (PCR amplification and sequencing). This substitution allows for the resolution of hundreds of antibodies by leveraging the high specificity and sensitivity of nucleic acid amplification and sequencing technologies.
2Quantity of substance
If mass spectrometry is used for proteomic detection, then it can detect proteins, but it requires expensive instrumentation, is heavily biased towards abundant protein species, and produces only semi-quantitative data
Solution Approach 1:
The patent uses oligonucleotide tags as intermediaries attached to antibodies that bind to target proteins. These tags serve as molecular barcodes that can be amplified and sequenced to provide precise quantification of protein abundance, replacing the semi-quantitative mass spectrometry approach with a highly quantitative molecular counting method.
Solution Approach 2:
The patent creates molecular copies of antibody-protein interactions through PCR amplification of the oligonucleotide tags. Each tag serves as a copyable molecular record of the binding event, allowing for precise quantification through counting amplified copies rather than relying on mass spectrometry signal intensities.
3Productivity
If CyTof (flow cytometry coupled to mass spectrometer) is used for single cell proteome detection, then it partially addresses throughput limitation, but it is extremely expensive, is not perfectly quantitative, and cannot be expanded beyond detection of about one hundred individual proteins
Solution Approach 1:
The patent replaces the expensive and complex CyTof system (combining flow cytometry and mass spectrometry) with a simpler, more cost-effective approach using oligonucleotide-tagged antibodies combined with PCR amplification and sequencing. This substitution maintains high throughput for single-cell analysis while dramatically reducing cost and instrument complexity.
Solution Approach 2:
The patent uses PCR amplification to create copies of oligonucleotide tags from single cells, enabling high-throughput analysis without requiring expensive mass spectrometry instrumentation. The copied molecular signals can be processed using standard sequencing technologies, expanding the number of detectable proteins beyond the one hundred protein limit of CyTof.
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 identification of a large number of proteins in single cells and populations with improved quantification and reduced bias, facilitating disease diagnosis and treatment monitoring.
Implementation Method 1
contacting a population of cells with multiple antibodies under conditions that promote specific binding of the antibodies to target antigens of the cells
Implementation Method 2
amplifying the unique molecular identifier sequences and antibody identifier sequences
Data Source
Figure 1A~1E
Figure 2A~2G
Figure 3
AI summary
Provided herein are compositions and methods for the identification of an expression profile in a single cell or population of cells. Kits for use with the disclosed methods are also provided, including antibodies, with a unique molecular identifier and antibody identifier, and primers for amplification of the antibody identifier sequence.