De Novo Protein Sequencing Using Dual-Protease Mass Ladders
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Solution Overview
Problem
Current protein sequencing methods, such as Edman degradation and mass spectrometry, are low-throughput and rely on incremental advancements, with limited progress in developing 'next-generation' platforms for global protein sequencing at the single amino acid residue level.
Innovation Solution
A method involving the use of two proteases, Trypsin and Tryp-N, that cleave peptide bonds before and after basic amino acids, respectively, combined with mass spectrometry to determine amino acid sequences by analyzing differences in peptide fragment ions, allowing for de novo sequencing without prior sequence information.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional Edman degradation or mass spectrometry methods are used for protein sequencing, then sequencing can be performed with established accuracy, but the throughput remains low and sequencing speed is limited
Solution Approach 1:
The patent segments the protein sequencing process by using multiple proteases (Trypsin and Tryp-N) to cleave peptide bonds at different positions (after and before basic amino acids respectively), generating complementary sets of peptide fragments that can be analyzed in parallel through mass spectrometry, thereby increasing throughput while maintaining accuracy
Solution Approach 2:
The patent performs preliminary enzymatic digestion of the protein with two different proteases before mass spectrometry analysis. This preliminary action creates overlapping peptide maps that facilitate de novo sequencing by providing multiple cleavage patterns, enabling faster and more accurate sequence determination without requiring prior sequence information
2Adaptability or versatility
If de novo sequencing is performed without prior sequence information, then novel protein sequences can be determined, but the process requires extensive fragmentation and analysis increasing complexity
Solution Approach 1:
The patent divides the complex de novo sequencing problem into manageable segments by using two proteases with complementary specificities. Trypsin cleaves after basic amino acids while Tryp-N cleaves before basic amino acids, creating overlapping peptide fragments that can be independently analyzed and then assembled, reducing the overall complexity of sequencing unknown proteins
Solution Approach 2:
The patent uses proteolytic peptides as intermediaries between the unknown protein sequence and the mass spectrometry detector. The dual-protease digestion strategy generates peptide fragments with known mass differences corresponding to specific amino acids, serving as mediators that enable de novo sequencing by bridging the gap between unknown protein sequences and identifiable mass spectral data
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 high-throughput de novo determination of amino acid sequences, improving sequencing speed, accuracy, and sensitivity, particularly for proteins like monoclonal antibodies, by leveraging the specific cleavage patterns and mass spectrometry analysis.
Implementation Method 1
contacting a first sample containing the polypeptide of interest with a first protease that cleaves peptide bonds after a basic amino acid... contacting a second sample containing the polypeptide of interest with second protease that cleaves peptide bonds before a basic amino acid
Implementation Method 2
determining masses of the first set of fragmented peptide ions... determining masses of the second set of fragmented peptide ions
Data Source
AI summary
A method for determining an amino acid sequence of a polypeptide, including comprising: contacting a first sample containing the polypeptide with a first protease (e.g., Trypsin) to produce a first set of digested peptide fragments; fragmenting the first set of digested peptide fragments to produce a first set of fragmented peptide ions; determining masses of the first set of fragmented peptide ions; contacting a second sample containing the polypeptide with a second protease (e.g., Tryp-N); fragmenting the second set of digested peptide fragments to produce a second set of fragmented peptide ions; selecting pairs of peptide ions from the first and the second set of fragmented peptide ions that differ in mass by a mass of an arginine amino acid residue or a lysine amino acid residue; assigning an ion type (either N-terminal peptide ion or C-terminal peptide ion) to the selected pairs of the peptide ions from two sets of fragmented peptide ions; selecting a mass ladder of the same-type peptide ions in either set of fragmented peptide ions with incremental mass by the mass of amino acid residue(s), and assembling the identified amino acid residues from the mass ladder to determine the amino acid sequence of the polypeptide of interest.