Protein Primary Structure Verification via Ion Cluster Comparison

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Solution Overview

Problem

Current methods for verifying the primary structure of proteins, particularly recombinant proteins, face challenges in accurately detecting structural variations and modifications due to limitations in high-performance liquid chromatography and mass spectrometry, especially for proteins with complex structures like monoclonal antibodies and glycoproteins.

Innovation Solution

A method involving high-resolution mass spectrometry that compares observed ion clusters from protein samples with simulated ion clusters generated based on the protein's chemical formula, allowing for the identification of the best-fit cluster to verify the protein's primary structure by removing modifications such as glycosylation and analyzing full-length proteins without enzymatic digestion.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If high-performance liquid chromatography and mass spectrometry are used to verify protein primary structure, then structural variations and modifications can be detected, but the detection accuracy is insufficient for proteins with complex structures like monoclonal antibodies and glycoproteins

Engineering Contradiction:

Improvedetection accuracy of structural variationsVSAvoiddifficulty in detecting modifications in complex proteins

Core Design Contradiction:

Measurement precisionVSDifficulty of detecting and measuring

Solution Approach 1:

The patent segments the complex protein analysis into distinct charge state components. By separating and analyzing ion clusters corresponding to different charge states individually, then integrating the results, the method achieves higher detection accuracy for structural variations and modifications in complex proteins like monoclonal antibodies and glycoproteins

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent introduces a new dimension of analysis by utilizing charge state as an additional parameter. Instead of analyzing all ions uniformly, the method categorizes ions by their charge states, creating a multi-dimensional analysis framework that enhances the detection of structural variations and post-translational modifications

Inventive Principle:

Principle #17Another dimension (Dimensionality change)

2Loss of information

If enzymatic digestion is used to analyze proteins, then peptide sequences can be obtained, but the full-length protein structure information is lost

Engineering Contradiction:

Improveinformation retention of full-length protein structureVSAvoidease of protein analysis process

Core Design Contradiction:

Loss of informationVSEase of manufacture

Solution Approach 1:

The patent creates a spectral copy or fingerprint of the full-length protein by analyzing intact protein ions. The integrated ion cluster pattern serves as a unique identifier that preserves complete structural information without requiring physical fragmentation, thereby maintaining full-length protein structure information while enabling comprehensive analysis

Inventive Principle:

Principle #26Copying

3Measurement precision

If modifications such as glycosylation are removed before analysis, then the protein core structure can be verified, but the complete primary structure including modifications cannot be confirmed

Engineering Contradiction:

Improveverification accuracy of complete primary structureVSAvoidcomplexity of sample preparation process

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent develops a universal analysis method that handles both modified and unmodified protein forms simultaneously. The integrated ion cluster approach can verify the protein core structure while also detecting and characterizing modifications like glycosylation in a single analysis run, eliminating the need for separate sample preparation steps

Inventive Principle:

Principle #6Universality (Multi-functionality)

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 precise verification of protein primary structures, including post-translational modifications, by integrating ion clusters across different charge states and comparing them with simulated clusters, thereby overcoming the limitations of existing analytical techniques.

Implementation Method 1

collecting mass spectra of the protein sample

Methodology Applied

Scientific EffectMass spectrometry:

Implementation Method 2

preparing a protein sample for mass spectrometric analyses

Methodology Applied

Scientific EffectIonization: Ionisation

Data Source

PatentUS11635437B2Method for verifying the primary structure of protein

Publication Date: 2023.04.25 SUN JET BIOTECH INC

AI summary

Disclosed herein is a method for verifying the primary structure of a protein through comparative analyses between ion clusters observed in mass spectra and a series of simulated ion clusters deduced from its putative chemical formula. The method comprises the steps of: preparing a protein sample for mass spectrometric analyses; collecting mass spectra of the protein sample; obtaining master ion cluster from a plurality of ion clusters in the mass spectra; producing a series of simulated ion clusters according to the chemical formula of the protein; finding the best fit for the master ion cluster among the series of simulated ion clusters; and verifying if said best-fit simulated ion cluster corresponds to the chemical formula of the protein.