Protein Quantification via Near Infrared Spectral Imaging
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Solution Overview
Problem
Current methods for protein and peptide quantification, especially after gel electrophoresis, are either expensive, time-consuming, destructive, or lack precision, and often require external contrast agents and extensive expertise.
Innovation Solution
A method based on near-infrared spectral imaging (NIRSI) that quantifies proteins by analyzing the inherent contrast from molecular vibrations in protein gel bands, eliminating the need for external contrast agents and simplifying the process while providing precise protein content calculations using calibration curves.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If amino acid analysis is used for protein quantification, then measurement precision is improved, but cost and processing time increase significantly
Solution Approach 1:
The patent extracts and utilizes the inherent infrared spectral information already present in the protein gel bands, eliminating the need for time-consuming hydrolysis, derivatization, and separation steps required by amino acid analysis. The infrared spectrum directly provides protein quantification data from the intact gel bands.
Solution Approach 2:
The protein samples themselves provide the measurement signal through their inherent infrared absorption properties. The amide I and amide II bands in the infrared spectrum arise from the protein's own molecular vibrations, requiring no external contrast agents or complex sample preparation.
2Measurement precision
If amino acid analysis is used for protein quantification, then measurement precision is improved, but operational complexity and cost increase
Solution Approach 1:
The patent removes the complex multi-step amino acid analysis procedure and replaces it with direct infrared spectral imaging of the gel bands. This extracts the essential measurement information while eliminating unnecessary complexity.
Solution Approach 2:
The patent replaces the mechanical and chemical manipulation steps of amino acid analysis (hydrolysis, derivatization, separation) with a non-destructive optical measurement using infrared spectral imaging, significantly simplifying the operational procedure.
3Ease of operation
If UV absorbance method is used for protein quantification, then ease of operation is improved, but measurement precision deteriorates due to gel interference
Solution Approach 1:
The patent transitions from UV absorbance measurement (which is blocked by the gel matrix) to infrared spectral imaging, operating in a different spectral dimension where the gel is transparent and protein bands exhibit characteristic absorption bands for quantification.
4Difficulty of detecting and measuring
If external contrast agents are used for protein detection in gels, then detection capability is improved, but sample destruction and additional processing steps occur
Solution Approach 1:
The protein samples provide their own detection signal through inherent infrared absorption properties. The amide I and amide II bands arise from the protein's own molecular structure, eliminating the need for external contrast agents like stains or fluorescent labels.
Solution Approach 2:
The patent extracts and utilizes the intrinsic infrared spectral information from the proteins, removing the need for additional contrast agents and their associated processing steps.
5Measurement precision
If destructive analysis methods are used for protein quantification, then measurement precision is improved, but sample recovery is lost
Solution Approach 1:
The patent replaces destructive chemical analysis methods with non-destructive infrared spectral imaging, allowing the protein samples to remain intact in the gel for potential recovery and further analysis.
Solution Approach 2:
The infrared measurement process does not consume or alter the protein samples, as it relies on the samples' inherent spectral properties rather than chemical reactions that would destroy or modify them.
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 allows for fast, reliable, and precise protein quantification directly from gel electrophoresis without destroying the samples or requiring additional processing steps, achieving sensitivity down to 10 ng of protein content with reduced operational complexity and cost.
Implementation Method 1
acquiring an infrared spectral dataset from the at least one protein band using near infrared spectral imaging (NIRSI)
Implementation Method 2
The infrared spectrum recorded with a spectral resolution between 1 and 100 cm−1. In one embodiment, the infrared spectrum is recorded in a range between 12000 and 3000 cm−1
Data Source
AI summary
The present invention introduces a protein/peptide quantification method based on near infrared spectral imaging of protein gel bands obtained from gel electrophoresis methodologies. Spectral imaging is a fast and reliable method that is simple to use and easily applicable to several procedures. When proteins are separated through gel electrophoresis, the gel is imaged using a near infrared spectrometer and a standard curve is used to calculate the protein/peptide content based on its relative near infrared absorbance to standard proteins. Compared to other protein quantification techniques, the methods of the present invention quantifies the proteins separated by gel electrophoresis without the need for contrast reagents or for purifying protein out of the gel, shortening the processing time, reducing the number of steps involved, and eliminating the need for additional chemicals.