Single-Cell Proteomics Using Oligo-Tagged Antibody Sequencing
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Solution Overview
Problem
Current methods for proteomic detection in single cells and cell populations are expensive, biased towards abundant proteins, and lack multiplexed protein measurements, providing only semi-quantitative data.
Innovation Solution
A method involving contacting cells with multiple antibodies conjugated to oligonucleotides, separating cells into compartments, amplifying unique molecular and antibody identifier sequences, and analyzing these sequences to identify expression profiles, allowing for the quantification of multiple proteins in single cells or populations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If mass spectrometry is used for proteomic detection, then detection capability is provided, but the method is heavily biased towards abundant protein species and produces only semi-quantitative data
Solution Approach 1:
The patent introduces oligonucleotide tags as intermediaries between antibodies and detection systems. These tags carry unique molecular identifiers that enable accurate quantification through sequencing, while the antibody-antigen binding maintains specificity. This intermediary approach transfers the detection function from mass spectrometry to sequencing technology, resolving the contradiction between quantification accuracy and detection bias.
Solution Approach 2:
The patent replaces the mass spectrometry detection mechanism with a sequencing-based detection mechanism. Instead of measuring protein mass and abundance directly through physical/chemical properties, the system uses oligonucleotide tags that can be amplified and sequenced, providing digital, quantitative data that is not biased by protein abundance levels.
2Measurement precision
If antibody detection methods are used, then quantitative measurement is improved, but multiplexed protein measurements cannot be performed when fluorescence spectral signatures cannot be resolved
Solution Approach 1:
The patent transitions from spectral dimension (fluorescence wavelengths) to sequence dimension (oligonucleotide sequences). Instead of relying on fluorescent spectral signatures that become unresolved when mixing many antibodies, the system uses oligonucleotide tags with unique molecular identifiers that can be distinguished through high-throughput sequencing, enabling multiplexed detection of hundreds of proteins simultaneously.
Solution Approach 2:
The patent changes the detection parameter from fluorescence intensity and spectral signature to oligonucleotide sequence identity and molecular identifier counts. This parameter transformation allows for the resolution of many more distinct protein targets because sequencing can distinguish between vastly more unique sequences than fluorescence can distinguish between spectral signatures.
3Productivity
If flow cytometry coupled to mass spectrometer (CyTof) is used, then throughput limitation is partially addressed, but the method is extremely expensive and cannot be expanded beyond detection of about one hundred individual proteins
Solution Approach 1:
The patent extracts the detection function from the expensive mass spectrometer and replaces it with a sequencing-based approach. By removing the mass spectrometry component and using only antibody tagging with oligonucleotides followed by sequencing, the system achieves similar or better throughput at significantly lower cost while expanding detection capacity beyond one hundred proteins.
Solution Approach 2:
The patent uses oligonucleotide tags as informational copies of protein-antibody binding events. Instead of directly analyzing the protein or its complex interactions with expensive instrumentation, the system creates nucleic acid copies of the binding information that can be amplified and sequenced using inexpensive, high-throughput methods, thereby multiplying the detection capacity.
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 accurate, quantitative measurement of multiple proteins in single cells or populations, overcoming the limitations of existing technologies by providing high-throughput, cost-effective, and multiplexed protein detection.
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.