Hydrophobically Modified Enzymes for Cleaner Peptide Mapping
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Solution Overview
Problem
Chromatogram contamination by intact enzyme peaks during peptide mapping and other analytical techniques, particularly when using high stoichiometric ratios of autolysis-resistant proteases, masks peptide fragment peaks and complicates protein identification and characterization.
Innovation Solution
Hydrophobically modify enzymes to increase their retention time on reversed-phase chromatography columns, ensuring they elute later than the analyte fragments, thereby preventing chromatogram contamination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high stoichiometric ratios of autolysis-resistant proteases are used, then complete digestion of the protein of interest is achieved, but intact enzyme peaks coelute with peptide fragments contaminating the chromatogram
Solution Approach 1:
The patent modifies the hydrophobicity parameter of the enzyme by introducing hydrophobic amino acid residues at the C-terminus. This changes the enzyme's interaction with the reversed-phase chromatography stationary phase, increasing its retention time so that it elutes after the peptide fragments, thereby separating the enzyme peak from the analyte peaks and eliminating contamination while maintaining complete digestion capability
Solution Approach 2:
The patent segments the enzyme molecule by adding a distinct hydrophobic C-terminal tail that acts as a chromatographic tag. This segmented structure allows the enzyme to be retained on the column longer than the peptide fragments, effectively separating the enzyme from the digest products in the chromatogram
2Object-generated harmful factors
If low stoichiometric ratios of protease enzyme are used, then enzyme autolysis is reduced, but not all protein molecules are digested resulting in low intensity chromatographic peaks
Solution Approach 1:
The patent changes the hydrophobicity parameter of the enzyme to enable its separation from peptide fragments via reversed-phase chromatography. This allows the use of higher stoichiometric ratios (improving digestion completeness and peak intensity) while the chromatographic separation prevents enzyme peaks from contaminating the analyte chromatogram, thus maintaining measurement precision
3Reliability
If intact protease enzyme is present in the reaction mixture, then enzyme activity is maintained, but the enzyme peak masks less intense peptide fragment peaks
Solution Approach 1:
The patent modifies the enzyme's hydrophobicity by adding hydrophobic residues at the C-terminus, which changes its retention characteristics on reversed-phase chromatography columns. This parameter change ensures the enzyme elutes after all peptide fragments, allowing the enzyme to remain active in the reaction mixture while its peak does not mask any analyte peaks, thus maintaining both reliability and 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
Prevents intact enzyme peaks from overlapping with analyte fragments, enhancing the clarity and accuracy of chromatographic analysis.
Implementation Method 1
the at least one enzyme is hydrophobically modified to increase a retention time of the at least one enzyme such that the at least one enzyme elutes from the reversed-phase chromatography column later than the fragments of the analyte
Data Source
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AI summary
The present invention relates to a method for analyzing a sample, the method comprising: (a) incubating a sample comprising an analyte with at least one enzyme to produce a digestion mixture comprising fragments of the analyte; (b) loading the digestion mixture onto a reversed-phase chromatography column: and (c) performing reversed-phase chromatography on the digestion mixture, wherein the at least one enzyme is hydrophobically modified to increase a retention time of the at least one enzyme such that the at least one enzyme elutes from the reversed-phase chromatography column later than the fragments of the analyte.