Label-Free Single-Cell Glycan Profiling Platform
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Solution Overview
Problem
Current methods for single-cell glycomics are limited by the minute amounts of biological material available and the need for labeling in MS analysis, leading to sample losses and biased profiling, with no direct analysis, quantitation, and accurate structural characterization of glycans at the single-cell level.
Innovation Solution
An integrated platform combining online in-capillary sample processing with high-sensitivity label-free capillary electrophoresis-mass spectrometry for N-glycan profiling, using PNGase F to release glycans from glycoproteins without modification, allowing direct and unbiased characterization and quantification of single-cell surface N-glycomes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If labeling methods are used in MS analysis to increase detectability, then ionization efficiency and detectability of released glycans are improved, but sample losses occur during sample processing and profiling becomes biased
Solution Approach 1:
The patent extracts and analyzes glycans in their native, unlabeled state by directly infusing released glycans into the mass spectrometer. This eliminates the need for labeling reagents and the associated sample losses during labeling and cleanup steps, while maintaining detectability through optimized MS parameters and direct infusion techniques.
Solution Approach 2:
The patent introduces an online glycan release system that serves as an intermediary between sample preparation and MS analysis. Glycans are released from glycoproteins directly in the capillary and immediately analyzed without requiring labeling, thus eliminating sample losses associated with labeling procedures while maintaining structural integrity and detectability.
2Measurement precision
If labeling methods are used in MS analysis, then ionization efficiency is improved, but native structural features of glycans are modified and accurate structural characterization is compromised
Solution Approach 1:
The patent extracts and analyzes glycans in their native, unlabeled state by directly infusing released glycans into the mass spectrometer. This eliminates the need for labeling reagents that would modify glycan structures, thereby preserving native structural features including sialylation, fucosylation, and branching patterns for accurate structural characterization.
Solution Approach 2:
The patent optimizes mass spectrometry parameters (ionization voltage, collision energy, scan range) to achieve sufficient ionization efficiency and detectability of unlabeled glycans. By adjusting these parameters, the system maintains adequate signal intensity without requiring structural modification through labeling, thus preserving native glycan structures for accurate characterization.
3Measurement precision
If conventional MS-based methods are used for glycan analysis, then quantitation is achieved, but sample processing steps lead to sample losses and biased profiling
Solution Approach 1:
The patent merges sample preparation and analysis into a single online process. Glycans are released from glycoproteins directly in the capillary and immediately analyzed by mass spectrometry without requiring separate labeling and cleanup steps. This integrated approach eliminates multiple handling steps that cause sample losses while maintaining quantitation capability through direct measurement of released glycans.
Solution Approach 2:
The patent implements continuous online analysis where glycans are released and analyzed in an uninterrupted flow. The online capillary system maintains continuous flow of released glycans directly into the mass spectrometer, eliminating discrete processing steps that cause sample losses and enabling continuous quantitation without interruption or bias from batch processing.
4Loss of information
If single-cell analysis is performed to characterize distinct cell subpopulations, then biological insight is improved, but the minute amounts of biological material available limit the analysis
Solution Approach 1:
The patent enhances the sensitivity of the mass spectrometry system by optimizing parameters such as ionization voltage, collision energy, and scan range. These parameter adjustments enable detection and quantitation of glycans from minute amounts of biological material obtained from single cells, thereby preserving the ability to characterize distinct cell subpopulations despite limited sample quantity.
Solution Approach 2:
The patent replaces conventional bulk sample processing with a highly sensitive online capillary system that is specifically optimized for analyzing trace amounts of material. This substitution enables the system to handle the minute quantities of glycans released from single cells, preserving biological insight from rare cell subpopulations that would be undetectable using conventional methods.
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 detection of up to 100 N-glycans per single cell with direct and unbiased profiling, preserving native structural features and eliminating sample losses, providing accurate structural characterization and quantitation of N-glycans in single mammalian cells and limited biomedically-relevant samples.
Implementation Method 1
allowing the glycan release agent to release one or more glycan moieties from the glycoprotein or glycolipid without modification of glycan structure or composition
Implementation Method 2
separating the released glycan moieties within the capillary tube or channel using the open tube capillary electrophoresis instrument or the microfluidic capillary electrophoresis instrument based on charge and hydrodynamic volume of the released glycan moieties
Implementation Method 3
injecting the separated glycan moieties from an outlet of the capillary tube or channel into the mass spectrometer, whereby the separated glycan moieties are ionized and fragmented to form a plurality of charged glycan fragments
Data Source
AI summary
An integrated platform is provided for direct and unbiased label-free native analysis of N-glycans from single cells and biological samples as small as 1 nL or less. An in-capillary sample processing method is coupled with high-sensitivity label-free capillary electrophoresis and mass spectrometry. Direct, label-free characterization and quantification of single-cell surface N-glycomes can be performed with the detection of up to 100 N-glycans per single cell and up to 400 N-glycans per nL of blood. N-glycome alterations can be detected at the single-cell level for diagnosis of medical conditions. The platform also preserves cell integrity and therefore can be used for spatial glycomic and multiomic profiling at the single cell level.