Bathocuproine-Based Peptide Quantitation Assay
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Solution Overview
Problem
Current methods for determining peptide concentrations, such as UV absorption and BCA assays, are insensitive, consume large sample volumes, and lack reproducibility, making them unsuitable for precise analysis in mass spectrometry.
Innovation Solution
A direct peptide or protein concentration method using a reagent composition that forms a colored complex with a specific formula, measured at 450 nm to 500 nm, allowing for accurate quantitation with reduced sample volume and improved sensitivity, utilizing bathocuproinedisulfonic acid disodium salt hydrate and copper sulfate.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If UV absorption or BCA assays are used to determine peptide concentration, then the method is commercially available and relatively simple to perform, but the sensitivity is insufficient and sample volume consumption is excessive
Solution Approach 1:
The patent changes the detection parameters by using a different chemical reaction system (copper chelation with bathocuproine disulfonate) that operates at alkaline pH and produces a highly sensitive colorimetric signal. This parameter change enables detection with much lower sample volumes while achieving the required sensitivity for peptide quantification
Solution Approach 2:
The patent introduces copper ions and bathocuproine disulfonate as intermediary substances that mediate the detection process. The copper-peptide complex formation followed by bathocuproine chelation creates a highly sensitive detection system that requires minimal sample volume while providing accurate concentration measurements
2Reliability
If current protein quantitation methods are used, then the procedures are established and commercially available, but they lack reproducibility and sample characterization capability
Solution Approach 1:
The patent performs preliminary sample characterization through copper chelation analysis before mass spectrometry experiments. This preliminary action provides normalization data that improves reproducibility of subsequent MS experiments, establishing a standardized workflow that can be consistently applied across different samples and experiments
3Measurement precision
If biuret or Lowry methods are used, then protein quantitation can be performed, but the sensitivity is insufficient and interference from common reagents occurs
Solution Approach 1:
The patent changes the detection chemistry to use copper chelation with bathocuproine disulfonate at alkaline pH, which has different selectivity characteristics compared to biuret or Lowry methods. This parameter change reduces interference from common reagents like detergents while enhancing sensitivity for peptide detection
Solution Approach 2:
The patent converts the alkaline conditions that might be considered harsh into a benefit, as the alkaline pH environment enhances the copper-chelate colorimetric reaction sensitivity and selectivity, while being compatible with many peptide samples and reducing interference from certain reagents
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
The method provides enhanced sensitivity and reduced variability, enabling robust signal detection with a high signal-to-noise ratio, allowing for precise quantitation of peptides in small volumes, suitable for mass spectrometry analysis.
Implementation Method 1
combining the sample with a quantitation assay reagent composition comprising a complex to form a mixture where the complex comprises the following general formula... measuring absorbance of the colored complex at 450 nm to 500 nm
Implementation Method 2
measuring absorbance of the colored complex at 450 nm to 500 nm as a direct indicator of peptide or protein concentration
Data Source
AI summary
Peptide and/or protein quantitation methods, kits, and compositions, particularly useful for mass spectrometry, are provided herein based on a bathocuproine-based composition complex such as bathocuproinedisulfonic acid disodium salt hydrate complex. The methods are one-step rapid absorbance methods using small sample volumes. They produce a robust signal with high signal to background ratio and accurately quantitate even complex peptide mixtures with low variability and high sensitivity.