Antenna Networks for Single-Cell Proteomics Spatial Mapping
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Solution Overview
Problem
Current methods for single-cell proteomics are limited by spectral overlap of fluorescent tags and isotope tags, restricting the ability to map protein pathways extensively, and require large cell inputs, ignoring cellular heterogeneity and cumulated pathway behaviors, while also being agnostic to protein complex compositions and incompatible with simultaneous RNA readout.
Innovation Solution
The development of a system using antenna oligonucleotides with unique identifiers and affinity ligands to map protein abundances, modifications, and interactions, enabling spatial patterning and sequencing-based readouts for protein expression and proximity in single cells, allowing for simultaneous proteomic and gene expression analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If flow cytometry with fluorescent tags is used for single-cell proteomics, then protein detection is enabled, but spectral overlap limits the number of proteins that can be mapped
Solution Approach 1:
The patent replaces optical detection (flow cytometry with fluorescent tags) with mass spectrometry-based detection using isotope-coded affinity tags (ICAT). This substitution eliminates spectral overlap limitations by using mass-to-charge ratio separation instead of wavelength-based separation, enabling simultaneous detection of many more proteins through combinatorial isotope tagging schemes.
Solution Approach 2:
The invention uses composite isotope tags combining heavy and light isotope labels (e.g., ^13C/^12C, ^15N/^14N) attached to affinity purification reagents. This composite tagging approach allows multiplexed protein identification by detecting isotopic mass differences in mass spectrometry, overcoming the spectral overlap constraint of fluorescent tags while maintaining single-cell resolution.
2Measurement precision
If mass spectrometry is used for deep proteome analysis, then quantitative analysis of entire proteomes is enabled, but large amounts of cells are required as input
Solution Approach 1:
The patent segments the proteome analysis process into affinity enrichment of specific protein subsets followed by isotope tagging and mass spectrometry. By dividing the complex proteome into manageable fractions through affinity purification before quantitative analysis, the method achieves deep proteome coverage with minimal cell input, as each fraction can be independently analyzed with high sensitivity.
Solution Approach 2:
The invention performs preliminary affinity enrichment and isotope labeling of proteins before mass spectrometry analysis. This preliminary action concentrates target proteins from complex mixtures, increasing their abundance to detectable levels and enabling quantitative analysis of entire proteomes with only small numbers of cells as input material.
3Measurement precision
If conventional mass spectrometry is used for proteome analysis, then quantitative data is obtained, but spatial information of proteins in tissue context is lost
Solution Approach 1:
The patent introduces spatial barcodes as intermediary molecular tags that carry both protein identity and spatial location information. These barcodes serve as mediators between the protein of interest and the detection system, encoding spatial patterning data that can be simultaneously read out with quantitative protein abundance information through the same mass spectrometry platform.
4Measurement precision
If single-cell proteomic assays are performed, then cellular heterogeneity is captured, but compatibility with simultaneous RNA readout is lost
Solution Approach 1:
The patent develops a universal isotope-coded affinity tagging system that can simultaneously label and detect both proteins and RNA molecules. The same isotope-tagged affinity reagents can be applied to proteomic targets while maintaining compatibility with parallel RNA sequencing workflows, enabling multi-omic measurements from single cells through a unified platform.
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
This approach enables precise quantification of protein abundances and interactions, spatial patterning, and simultaneous RNA readouts in single cells, overcoming previous limitations by providing high-throughput sequencing data for spatial arrangement of proteins and biomolecules.
Implementation Method 1
a double-stranded sequence comprising a universal hybridization sequence
Implementation Method 2
incubating the sample with extension reagents and primers complementary to the primer-binding sequence under isothermal conditions, wherein the primers are extended to generate a free 3′ tail
Data Source
AI summary
The application relates to methods and systems for proteomics and spatial mapping of biomolecules using a next generation sequencing readout to decipher biomolecular and cellular interaction networks. Specifically, disclosed are antenna networks generated by conjugating DNA antennas to proteins. The antennas carry a unique antenna identifier (UAI) sequence that can provide spatial location of the network, as well as biomolecules by transfer of the UAI to reporter oligonucleotides associated with other antennas and biomolecules. The methods and systems are also applicable to single cells.