Serum Protease Digestion for Trace Protein Mass Spectrometry
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Solution Overview
Problem
Current methods for detecting proteins in serum or plasma samples using mass spectrometry face challenges in efficiently detecting trace proteins due to the dominance of abundant proteins like albumin, which leads to saturation and difficulty in identifying peptides of interest, requiring complex and costly pretreatment processes.
Innovation Solution
A method involving the direct addition of a protease to serum or plasma under non-denaturing conditions to prevent albumin digestion, allowing for specific digestion and enrichment of peptides of interest without the need for extensive pretreatment, such as denaturation or alkylation, thereby reducing the concentration range and enhancing the detection of trace proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional pretreatment methods (denaturation, reduction, alkylation) are used before protease digestion, then protein fragmentation efficiency is improved, but process complexity and cost increase
Solution Approach 1:
The invention performs protease digestion directly on native proteins without preliminary denaturation, reduction, or alkylation steps. The protease selectively digests trace proteins while leaving abundant proteins like albumin intact, achieving efficient fragmentation without complex pretreatment
Solution Approach 2:
The invention changes the digestion conditions by performing proteolysis under native conditions rather than denaturing conditions. This parameter change allows selective digestion of trace proteins while maintaining abundant proteins in their native state, simplifying the overall process
2Productivity
If protease digestion is performed under denaturing conditions, then protein fragmentation is enhanced, but albumin is also digested leading to saturation of the detection system
Solution Approach 1:
The invention creates different states for different proteins in the same sample. Abundant proteins like albumin remain in native state and are not digested, while trace proteins are selectively digested by the protease, achieving local differentiation in digestion behavior
Solution Approach 2:
Instead of digesting all proteins and then separating trace protein peptides from abundant protein peptides, the invention inverts the approach by preventing digestion of abundant proteins altogether, allowing direct detection of trace protein peptides without saturation
3Measurement precision
If complex pretreatment processes are used to remove abundant proteins, then detection sensitivity for trace proteins is improved, but time and cost increase
Solution Approach 1:
The invention extracts or removes the need for complex pretreatment steps by performing selective digestion directly on the native protein mixture. The protease naturally selectively acts on trace proteins while ignoring abundant proteins, eliminating the need for time-consuming removal steps
Solution Approach 2:
The protease performs self-service selective digestion based on the inherent properties of the proteins in the sample. The enzyme automatically distinguishes between trace and abundant proteins and digests only the trace proteins, without requiring external intervention or complex pretreatment
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 convenient and high-throughput quantitative determination of trace proteins by minimizing albumin-derived peptide detection, improving sensitivity, and reducing the time and cost associated with pretreatment processes, allowing for efficient detection of proteins of interest in a clinical setting.
Implementation Method 1
a method involving the direct addition of a protease to serum or plasma under non-denaturing conditions to prevent albumin digestion, allowing for specific digestion and enrichment of peptides of interest
Implementation Method 2
proteins are ionized by matrix-assisted laser desorption ionization or electrospray ionization (ESI)
Implementation Method 3
proteins are ionized by matrix-assisted laser desorption ionization
Data Source
AI summary
The present invention provides: a method of pretreating a serum or plasma sample for detection of a protein or a plurality of proteins of interest in a serum or plasma sample via mass spectrometry and a method of detecting such a protein or proteins, wherein proteins such as albumin present in abundance in a sample are removed in a convenient manner, thereby making it possible to collect digested peptides from the protein of interest. Specifically, the present invention provides a method of pretreating a sample for detecting proteins in a serum or plasma sample via mass spectrometry, comprising a step of adding a protease to the sample under non-denaturing conditions to digest proteins and a step of separating the obtained peptides from undigested proteins, and a method of detecting proteins, comprising subjecting the obtained peptides to mass spectrometry.