Partial Proteolytic Digestion for Rapid HbA1c Mass Spectrometry
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Solution Overview
Problem
Current methods for determining glycated hemoglobin A1c (HbA1c) using enzymatic digestion are time-consuming and not suitable for high-throughput measurements due to the need for overnight incubation and complex separation processes.
Innovation Solution
A method involving partial proteolytic digestion of HbA β-chain molecules, where 1% to 20% of the molecules are digested, followed by enrichment and analysis using mass spectrometry to rapidly and reliably determine HbA1c levels, with the use of a kit containing enzymes, buffers, and enrichment means.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional enzymatic digestion methods are used for HbA1c determination, then sufficient cleavage products are obtained for stable measurement signals, but the measurement process becomes time-consuming and unsuitable for high-throughput analysis
Solution Approach 1:
The patent applies partial proteolytic digestion by controlling the enzymatic reaction to digest only 5-50% of HbA1c molecules instead of complete digestion. This partial action generates sufficient peptide fragments for reliable MS detection while dramatically reducing the required incubation time from overnight to minutes or hours, thereby resolving the contradiction between measurement reliability and throughput
Solution Approach 2:
The patent changes key parameters of the enzymatic digestion process including enzyme-to-substrate ratio, incubation time, and temperature to optimize for partial digestion. By adjusting these parameters, the method achieves rapid peptide generation with adequate signal stability for MS analysis, enabling high-throughput processing without sacrificing measurement reliability
2Measurement precision
If complete proteolytic digestion is performed to ensure sufficient peptide fragments, then measurement signals are stable, but the digestion time increases to overnight incubation
Solution Approach 1:
The method deliberately performs incomplete digestion (5-50% of HbA1c molecules) instead of complete digestion. This partial action is sufficient to generate enough peptide fragments for stable MS signals while reducing digestion time from overnight to minutes or hours, effectively resolving the time-precision contradiction
Solution Approach 2:
The patent employs preliminary enrichment steps before MS analysis to concentrate the peptide fragments generated from partial digestion. This preliminary action compensates for the lower yield from partial digestion, ensuring sufficient signal stability without requiring extended digestion times
3Measurement precision
If conventional separation methods are used to separate complex peptide mixtures, then accurate MS detection is achieved, but the overall process becomes too slow for high-throughput measurements
Solution Approach 1:
The patent extracts and enriches specific peptide fragments of interest from the complex digestion mixture before MS analysis. This extraction step isolates the relevant analytes from interfering substances, maintaining detection accuracy while reducing the complexity and time of subsequent analysis, thereby enabling high-throughput processing
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 allows for accurate and rapid determination of HbA1c with a coefficient of variation of 2.5% or less, enabling efficient high-throughput analysis of patient samples.
Implementation Method 1
subjecting the sample from step (a) to a partial proteolytic digestion step wherein N-terminal fragments of hemoglobin A β-chain molecules are generated
Implementation Method 2
subjecting the sample comprising N-terminal fragments of hemoglobin A β-chain molecules to an analysis by mass-spectrometry (MS) wherein the amount or concentration of HbA1c in the sample is determined
Data Source
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AI summary
The present invention refers to a method for determining peptide fragments of glycated hemoglobin A (HbA1c) molecules by mass spectrometry (MS), a reagent kit, and a clinical diagnostic system adapted for performing the method.