Peptide Mass Tags for Native MS Protein Identification
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Solution Overview
Problem
Current native mass spectrometry techniques face inefficiencies in fragment ion generation from intact proteins, particularly in complex samples, leading to challenges in unambiguous protein identification and quantitation, especially for low-abundance proteins in mixtures.
Innovation Solution
A peptide mass tag with fragmentation propensity and a binding moiety is developed, allowing for selective fragmentation and recovery of target proteins, enabling unambiguous identification and multiplexing in native MS and tandem MS studies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If native mass spectrometry is used to study endogenous proteins directly from native environments, then structural and functional information is preserved, but fragment ion generation efficiency is insufficient for unambiguous protein identification
Solution Approach 1:
The invention introduces a peptide mass tag that segments the protein analysis process into two distinct phases: intact protein analysis for structural information and targeted fragment ion generation for identification. The tag contains a specific cleavage site that separates the protein into fragments suitable for database searching, resolving the contradiction between preserving native structure and generating identification fragments.
Solution Approach 2:
The peptide mass tag acts as an intermediary element that bridges native mass spectrometry and tandem mass spectrometry. It allows intact protein analysis to proceed in native conditions while providing a controlled fragmentation mechanism through the tag's cleavage site, enabling both structural preservation and identification fragment generation.
2Measurement precision
If tandem mass spectrometry is applied to complex samples with many proteins, then molecular identification is possible, but statistical likelihood of similar fragment weights increases leading to ambiguous identification
Solution Approach 1:
The invention applies local quality by focusing fragment ion generation on a specific region—the peptide mass tag—rather than the entire protein. This localized approach ensures that fragments originate from a defined sequence with unique characteristics, improving discrimination between different proteins in complex samples and reducing ambiguous identifications.
Solution Approach 2:
The peptide mass tag is designed with a predetermined cleavage site that ensures consistent and reproducible fragmentation at a specific location. This preliminary design of the fragmentation pattern allows for reliable database searching and reduces the statistical likelihood of false identifications by ensuring unique fragment weights.
3Stability of the object's composition
If intact protein fragmentation is performed in native mass spectrometry, then structural information is maintained, but fragment ion generation is inherently inefficient
Solution Approach 1:
The invention extracts the fragmentation function from the native protein structure by introducing a separate peptide mass tag with a dedicated cleavage site. The native protein structure remains intact and stable, while the tag provides the necessary fragmentation capability, effectively separating the structural preservation function from the fragment ion generation function.
4Ease of manufacture
If only terminal fragments are considered in database searching, then identification process is simplified, but ability to identify target protein with high confidence is limited
Solution Approach 1:
The peptide mass tag creates a defined segmentation point in the protein sequence, generating both N-terminal and C-terminal fragments relative to the cleavage site. This segmentation enables the use of internal fragments in addition to terminal fragments for database searching, improving identification confidence while maintaining a relatively simple analytical approach.
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 peptide mass tag provides diagnostic fragment ions for unambiguous protein identification and enables multiplexing, allowing for relative quantitation in pooled samples without disrupting initial molecular information.
Implementation Method 1
a peptide mass tag with fragmentation propensity and a binding moiety is developed, allowing for selective fragmentation and recovery of target proteins
Implementation Method 2
A plurality of such tags may be provided for multiplex applications, for example in a kit or library. The invention also relates to a polypeptide comprising a target protein or an affinity ligand for a target protein fused to said tag
Data Source
AI summary
The invention relates to a peptide mass tag for recovery and identification of target proteins by mass spectrometry. The invention further relates to fusion polypeptides including the peptide mass tag and a method of analysing a target protein comprising use of the peptide mass tag.