LC-MS/MS Protein Digestion Analysis via Segmented Intestinal Sampling
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Solution Overview
Problem
Current methods for measuring in vivo protein digestibility, such as fecal nitrogen balance experiments, are complex, time-consuming, and do not accurately reflect protein digestion and absorption in the body, lacking a comprehensive understanding of protein metabolism.
Innovation Solution
A method utilizing LC-MS-MS technique to analyze protein digestion and absorption by collecting intestinal contents, isolating proteins, digesting and desalting samples, and performing tandem mass spectrometry with database searching to identify dietary, host, and microbial protein sources, providing insights into protein digestion and metabolism.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fecal nitrogen balance experiment is used to measure in vivo protein digestibility, then measurement can be performed, but the operation is complicated and time-consuming
Solution Approach 1:
The patent replaces the traditional fecal nitrogen balance experiment (chemical analysis method) with mass spectrometry technology. The MS system directly identifies and quantifies proteins and peptides in intestinal contents, eliminating the need for complex nitrogen balance calculations and multiple correction factors, thereby simplifying the measurement process while maintaining accuracy.
Solution Approach 2:
The patent uses stable isotope-labeled proteins as internal standards and creates spectral libraries of digestive products. These copies serve as reference benchmarks that enable direct comparison and quantification, replacing the indirect nitrogen balance approach with a more direct and simplified measurement system.
2Measurement precision
If fecal nitrogen balance experiment is used, then protein digestibility can be measured, but it is long in time
Solution Approach 1:
The mass spectrometry system continuously analyzes protein and peptide samples from different intestinal segments, providing real-time data on digestion progression. This continuous analysis eliminates the intermittent, time-consuming nature of traditional nitrogen balance measurements and enables faster assessment of protein digestibility.
Solution Approach 2:
The patent performs preliminary database construction and spectral library preparation beforehand, containing pre-established reference data for rapid comparison during actual measurements. This preliminary work enables subsequent measurements to be completed much faster without sacrificing accuracy.
3Measurement precision
If traditional nitrogen balance method is used, then measurement can be performed, but it lacks in-depth understanding of digestion, absorption and metabolism
Solution Approach 1:
The patent divides the intestinal tract into multiple segments and analyzes protein digestion products in each segment separately using mass spectrometry. This segmentation reveals the progressive breakdown of proteins at different stages, providing detailed information about digestion processes that the bulk nitrogen balance method cannot detect.
Solution Approach 2:
The patent uses stable isotope labeling (different mass tags) to distinguish between dietary proteins, host proteins, and microbial proteins. This isotopic 'coloring' allows the MS system to track and differentiate various protein sources and their fates throughout digestion, providing comprehensive metabolic information that was previously lost.
4Measurement precision
If fecal nitrogen balance experiment is used, then apparent digestibility can be calculated, but it does not truly reflect the digestion and absorption of proteins in the body
Solution Approach 1:
The mass spectrometry system directly detects and identifies actual protein and peptide molecules present in intestinal contents, allowing the system to self-verify digestion status without relying on indirect nitrogen balance calculations. This direct detection provides more reliable and accurate reflection of true protein digestion and absorption.
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 simplifies the evaluation of protein digestibility by identifying proteins and their digestion products in intestinal segments, offering a deeper understanding of protein digestion and metabolism, and providing a scientific basis for protein utilization.
Implementation Method 1
Mass spectrometry is the core technology in proteomics research, is the basic means for protein identification, and can accurately measure the relative molecular mass, amino acid sequence and post-translational modification of peptides and proteins
Implementation Method 2
the peptides separated by the LC are ionized and charged by an external electric field before entering the MS
Implementation Method 3
liquid chromatography-mass spectrometry has become the mainstream method for high-throughput analysis of proteins
Implementation Method 4
The databases are compared by the Maxquant software and the protein databases of three sources (host, diet, and microorganisms) are compared to collect all the information on digestion, absorption and conversion of proteins in vivo
Data Source
AI summary
Disclosed is a method for evaluating in vivo protein nutrition based on an LC-MS-MS technique, including the following steps: (1) collecting contents from different intestinal segments, and extracting and isolating protein ingredients; (2) determining the concentration of proteins; (3) treating before carrying out mass spectrometry: including digestion and desalting of a whole protein solution; (4) LC-MS-MS analysis; (5) database searching; and (6) data processing. Proteomic technology is used to identify proteins in the contents of different intestinal segments and digestive products thereof, and the source of the proteins in the contents of different intestinal segments and the contents thereof can be determined therefrom. Through bioinformatic analysis, the function of differential proteins in the body can be further understood, where the gene expression of enzymes related to protein digestion and metabolism may be different, thereby providing a scientific basis for further scientific evaluation of protein digestion and utilization.