Intact Biomolecule Quantitation Using Short-Column LC-MS
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Solution Overview
Problem
Current methods for quantifying intact proteins using ligand binding assays or bottom-up liquid chromatography-mass spectrometry fail to provide structural information and are hindered by challenges in achieving good resolution and high protein recovery without loss to the stationary phase, along with long run times detrimental to high throughput analysis.
Innovation Solution
The use of a short column device with optimized stationary and mobile phases, featuring sorbent particles with specific dimensions and pore sizes, coupled with high-resolution mass spectrometry and temperature control, to enhance chromatographic separation and mass spectrometric sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If traditional ligand binding assays or bottom-up LC-MS are used for protein quantitation, then quantitation can be achieved, but structural information is lost and metabolic transformation cannot be determined
Solution Approach 1:
The patent segments the analysis approach by using targeted MS/MS detection of specific peptide fragments while maintaining intact protein context, allowing both structural information preservation and accurate quantitation through selective monitoring of diagnostic peptides
Solution Approach 2:
The patent uses peptide fragments as intermediaries that retain structural information from the parent protein while being suitable for MS detection and quantitation, bridging the gap between intact protein analysis and peptide-level detection
2Loss of information
If RPLC-HRMS is used for intact protein analysis, then structural information and complete quantitation picture are obtained, but resolution and protein recovery are compromised due to stationary phase interactions
Solution Approach 1:
The patent extracts only the essential information needed for quantitation by using targeted MS/MS detection of specific peptide ions, avoiding the need for extensive intact protein separation and reducing stationary phase interactions while maintaining structural information
Solution Approach 2:
The patent changes the detection parameters by using targeted mass spectrometry with specific m/z transitions instead of full-scan HRMS, reducing the time proteins spend in the chromatographic system and minimizing stationary phase interactions that cause loss
3Measurement precision
If optimized LC-MS methods are used for intact protein quantitation, then sensitivity is improved, but run times are extended which reduces throughput
Solution Approach 1:
The patent uses periodic action by implementing targeted MS/MS detection cycles that rapidly switch between multiple predefined transitions, achieving high sensitivity for multiple proteins in sequence without requiring long gradient separations for each
Solution Approach 2:
The patent maintains continuity of useful action by using continuous flow analysis with rapid targeted detection, eliminating the need for repeated column equilibration and re-optimization between samples, thereby maintaining high throughput while achieving sensitivity
4Manufacturing precision
If longer chromatographic columns are used to improve resolution, then separation quality increases, but run times are extended and throughput decreases
Solution Approach 1:
The patent extracts only the critical separation information by using targeted MS/MS detection of specific peptide fragments that provide sufficient resolution for quantitation without requiring long chromatographic columns, achieving both resolution and throughput
Solution Approach 2:
The patent applies local quality by focusing detection resources on specific diagnostic peptide transitions that provide the necessary resolution for each protein, rather than requiring uniform high resolution across the entire chromatographic run
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 improved protein recovery, higher throughput, and enhanced mass spectrometric sensitivity, allowing for more accurate and efficient quantitation of intact proteins with reduced sample preparation times and increased detection capabilities.
Implementation Method 1
chromatographic separation devices that comprise (a) sorbent particles having width ranging from 0.5 to 100 μm... wherein a direction of fluid flow from the inlet to the outlet of the housing is along the length of the volume of the sorbent particles
Implementation Method 2
methods useful for quantitation of biomolecules such as intact proteins and nucleic acids... linear mass spectrometric detection across a wide dynamic range with enhanced sensitivity
Data Source
AI summary
The present disclosure provides devices, systems, kits and methods useful for quantitation of biomolecules such as intact proteins and nucleic acids.