HDL Protein Extraction via Organic Acid Precipitation
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Solution Overview
Problem
Current methods for isolating high-density lipoproteins (HDL) are time-consuming and inefficient, requiring prolonged ultracentrifugation steps and multiple density adjustments, limiting throughput and making it challenging to detect and quantify HDL-associated proteins like ApoA1, especially when they are present in low concentrations.
Innovation Solution
A method involving the use of a strong organic acid and hydrophilic organic solvent to precipitate a sample, followed by centrifugation and separation with a non-polar solvent, allowing for the detection of HDL-associated proteins through a detection assay, such as mass spectrometry, which can be performed in a more rapid and parallel fashion, reducing interference from abundant proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If sequential density ultracentrifugation is used to isolate HDL, then HDL purification is achieved, but the process requires prolonged time (18-21 hours per step) and multiple density adjustments
Solution Approach 1:
The patent changes the physical-chemical parameters of the separation process by using organic acid precipitants (trifluoroacetic acid, formic acid, acetic acid) combined with organic solvents (acetonitrile, methanol, ethanol, propanol, isopropanol, butanol, tetrahydrofuran) to alter protein solubility and density characteristics, enabling HDL separation from other plasma proteins without requiring prolonged ultracentrifugation steps
Solution Approach 2:
The patent replaces the mechanical ultracentrifugation system with a chemical precipitation system using organic acids and solvents that selectively precipitate HDL-associated proteins, eliminating the need for prolonged mechanical separation and reducing isolation time significantly
2Reliability
If traditional ultracentrifugation methods are used, then HDL can be isolated, but throughput is limited due to prolonged processing time
Solution Approach 1:
The patent extracts and removes interfering proteins from the plasma sample using organic acid precipitation, selectively isolating HDL-associated proteins in the aqueous layer while removing other plasma proteins in the organic solvent layer, thereby enabling rapid processing and high throughput
3Quantity of substance
If abundant proteins are present in the sample, then complete protein extraction is achieved, but detection sensitivity for low-level modified amino acids is reduced
Solution Approach 1:
The patent extracts and removes interfering abundant proteins from the sample using organic acid precipitation, selectively isolating HDL-associated proteins in the aqueous layer while removing other plasma proteins in the organic solvent layer, thereby enabling rapid processing and high throughput
Solution Approach 2:
The patent applies local quality by using specific organic acids and solvents that selectively interact with HDL-associated proteins based on their unique chemical properties, allowing differential extraction and purification that enhances detection sensitivity for modified amino acids
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 method significantly reduces the time required for HDL-associated protein extraction and detection, enabling the efficient quantification of low-level modified amino acids like chlorinated tyrosine 192 and 2-hydroxytryptophan 72 in ApoA1, improving assay sensitivity and throughput compared to traditional ultracentrifugation methods.
Implementation Method 1
a strong organic acid and hydrophilic organic solvent are mixed with the sample; after centrifugation, the supernatant is transferred
Implementation Method 2
exposing the first mixed sample to centrifugal force such that the first mixed sample separates into a pellet and supernatant
Implementation Method 3
adding a non-polar organic solvent to the second container at a ratio of greater than 1:1 to generate a second mixed sample; exposing the second mixed sample to centrifugal force such that the second mixed sample separates into an upper organic solvent layer and a bottom aqueous layer
Data Source
AI summary
Provided herein are compositions, systems, and methods for extracting and detecting at least one HDL-associated protein (e.g., ApoA1) from a sample (e.g., plasma or serum sample). In certain embodiments, a strong organic acid and hydrophilic organic solvent are mixed with the sample; after centrifugation, the supernatant is transferred to a second container where it is mixed with a non-polar organic solvent; after centrifugation, the lower aqueous layer is transferred to a third container; and then at least a portion of the transferred aqueous layer is subjected to a detection assay such that at least one HDL-associated protein is detected.