Mixed-Phase SPE Material for Peptide Purification
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Solution Overview
Problem
Current methods for purifying peptides and polypeptides are inadequate in removing contaminants such as salts, fats, lipids, sugars, and interfering ions from biological samples, which can lead to system failures and poor performance in LC-MS measurements, especially in clinically relevant samples like blood plasma and urine.
Innovation Solution
A method involving loading samples onto mixed-phase solid phase extraction (SPE) material under acidic or neutral conditions, followed by washing with a composition comprising at least 50% organic solvent, and eluting with an alkaline composition containing at least 50% organic solvent to effectively remove contaminants and achieve high purity levels of peptides and polypeptides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional purification methods are used, then sample processing is simpler, but purification effectiveness is insufficient and contaminants remain
Solution Approach 1:
The patent employs mixed-phase SPE material combining reversed phase and ion exchange properties in a single composite medium. This composite approach enables simultaneous removal of diverse contaminants (salts, lipids, sugars, interfering ions) through multiple mechanisms, achieving high purification effectiveness (60-99% purity) while maintaining a relatively simple single-step process configuration
Solution Approach 2:
The method utilizes pH parameter changes to control the binding and elution of peptides. Loading is performed under acidic or neutral conditions where peptides bind to the mixed-phase material, then elution is achieved by switching to alkaline conditions. This parameter change enables effective separation and purification while maintaining process simplicity
2Object-affected harmful factors
If protein precipitation steps are used to reduce contaminants, then interfering ions are reduced, but sample loss increases due to poor peptide precipitation
Solution Approach 1:
The patent changes the chemical environment parameters (pH, ionic strength, organic solvent content) to favor peptide binding to the SPE material while contaminants remain in solution. During loading under acidic/neutral conditions, peptides bind selectively to the mixed-phase material while salts, lipids, and sugars are washed away. This parameter-controlled approach removes interfering ions without precipitating peptides, eliminating sample loss
Solution Approach 2:
The method extracts and removes contaminants (salts, lipids, sugars, interfering ions) from the sample matrix through the mixed-phase SPE material while retaining the peptides of interest. The washing steps selectively extract harmful substances without co-precipitating peptides, achieving contaminant removal without sample loss
3Ease of operation
If LC-MS incompatible detergents are used during sample handling, then ease of operation increases, but system reliability decreases due to instrument damage and performance loss
Solution Approach 1:
The mixed-phase SPE method extracts and removes detergents and other contaminating substances from the sample before LC-MS analysis. The washing steps with acidic or neutral compositions containing organic solvents effectively eliminate LC-MS incompatible detergents, protecting the instrument while maintaining ease of sample handling during preparation
Solution Approach 2:
The purification method performs preliminary removal of harmful substances (detergents, salts, lipids) before the critical LC-MS analysis step. By pre-cleaning the sample through mixed-phase SPE, the method prevents instrument damage and performance degradation, ensuring reliability while allowing flexible sample handling during preparation
4Productivity
If samples with high contaminant content are analyzed directly, then productivity is higher, but measurement precision deteriorates due to flooded mass spectrometer and poor signal quality
Solution Approach 1:
The method extracts and removes contaminants (salts, lipids, sugars, interfering ions) that would otherwise flood the mass spectrometer and degrade measurement quality. The mixed-phase SPE material selectively binds peptides while allowing contaminants to be washed away, ensuring high measurement precision without sacrificing analysis throughput
Solution Approach 2:
The method changes the chemical parameters of the sample matrix through controlled loading, washing, and elution steps. By adjusting pH and organic solvent content during these steps, the method optimizes peptide binding and contaminant removal, achieving both high productivity and excellent measurement precision
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 achieves purification levels of at least 60% and up to 99% purity, significantly improving the quality of samples and reducing system failures, allowing for more efficient downstream processes like LC-MS analysis without interference from contaminants.
Implementation Method 1
said material consists of or comprises reversed phase/ion exchange material
Implementation Method 2
said material consists of or comprises reversed phase/ion exchange material
Implementation Method 3
washing said mixed-phase SPE material with (ba) an acidic or neutral composition comprising at least 50% (v/v) organic solvent
Implementation Method 4
eluting said peptides and/or polypeptides from said mixed-phase SPE material with an alkaline composition comprising at least 50% (v/v) organic solvent
Data Source
AI summary
The present invention relates to a method of purifying peptides and/or polypeptides, said method comprising or consisting of: (a) loading a sample comprising peptides and/or polypeptides under acidic or neutral aqueous conditions on mixed-phase solid phase extraction (SPE) material, wherein said material consists of or comprises reversed phase/ion exchange material; (b) washing said mixed-phase SPE material with (ba) an acidic or neutral composition comprising at least 50% (v/v) organic solvent; and/or (bb) an acidic or neutral aqueous solution; and (c) eluting said peptides and/or polypeptides from said mixed-phase SPE material with an alkaline composition comprising at least 50% (v/v) organic solvent.