Protein De Novo Sequencing and Mutation Analysis

Protein molecules play a crucial role in the development of test kits, diagnostic reagents, and drug research. The primary structure of protein molecules is the core of their biological function. Therefore, accurate and rapid analysis of the complete protein sequence aids in the functional study of unknown proteins and is of great significance for the development and production of protein-based drugs, including monoclonal antibodies.Monoclonal antibodiesTraditional protein sequence analysis techniques, whether based on MALDI-TOF mass spectrometry or nanoLC-MS/MS platforms, require the use of a sequence database containing the identified protein during the identification process. The molecular weight data measured by mass spectrometry are compared with the molecular weight data obtained from the theoretical sequence fragments in the database to achieve protein sequencing and identification.

In actual protein sequence analysis, much protein information is not included in existing databases, such as a completely new protein not yet reported or a protein purified from a new species. In many cases, we can obtain partial theoretical data, but the missing part is crucial. For example, enzyme sequence modification information in commercially modified enzymes is a technical barrier for competitors intending reverse engineering because sequence changes can be varied (point mutations, deletions, insertions, replacements, modifications, fusions, etc.), resulting in thousands of possibilities without any direction to follow. Not only a specific enzyme in a test kit, but many protein drugs also need to be optimized through mutations to increase yield or stability. In many cases, protein sequences undergo mutations, and even a small sequence mutation in a drug can alter biological activity and trigger an immune response in the human body, leading to significant commercial losses. Based on the demand for unknown protein sequence and mutation analysis mentioned above, Biotech-Park has established a new generation of de novo sequencing and mutation analysis platform using high-resolution mass spectrometer Orbitrap Fusion Lumos and combining extensive bioinformatics analysis experience to achieve rapid and accurate analysis of the primary structure of proteins.

Protein de novo sequencing and mutation analysis process

 

Protein de novo sequencing and mutation analysis process

 

Platform advantages:

1. Complete sequence coverage (100% Sequence Coverage)
During protein sequence determination, we select five different proteases for enzymatic digestion, using the complementarity between different enzyme-digested peptides to achieve 100% complete sequence assembly of the protein molecule.
2. Advanced data processing algorithms
Based on literature published in Nature Biotechnology and professional proteomics journals like JPR, and combining existing professional software for database-independent protein sequence analysis, PEAKS Mass Spectrometry Software, we developed our method for processing raw mass spectrometry data. By adopting a broad input and narrow output principle, we achieve accurate identification of protein sequences without missing any valid data information.
3. High precision and accuracy
We use high-resolution and high-sensitivity Orbitrap Fusion Lumos mass spectrometer during sequencing. Compared to older generations of mass spectrometers (such as Orbitrap Elite, Q Exactive, etc.), Lumos improves the scanning speed of both primary and secondary mass spectrometry while ensuring the accuracy of peptide molecular weights. The increased primary mass spectrometry scanning speed helps the same peptide to be identified multiple times, enhancing the credibility of the results. The increased secondary mass spectrometry scanning speed allows better fragmentation (as shown in the figure below), ensuring result accuracy and enabling accurate determination of leucine and isoleucine through characteristic peaks of secondary fragmentation fragments.

 

Protein de novo sequencing and mutation analysis 2

4. Rigorous sequence prediction and strict sequence validation
We first perform sequence prediction on the sequencing results. Additionally, we build a new database with the determined sequence and adopt the Sequence Confirmation mode to re-analyze the collected mass spectrometry data. We only provide clients with completely matching results.

Application fields

1. In academic research, accurately determine the sequence information of unknown proteins or peptides according to research paper publishing needs;
2. Accurately determine the sequences of commercially modified proteins and enzymes. Many commercially valuable proteins (most of which are enzymes) have amino acid sequences that have been specifically modified, making the protein products have better characteristics than natural proteins. Since the exact genetic or amino acid sequences of these modified proteins are often not publicly available, and these protein products are easily obtainable, accurately determining the sequences of such protein products has great commercial value;
3. Accurately determine the sequence of proteins expressed by stable cell lines. Mutations (also known as sequence variants, SV) can easily occur during cell line construction and storage, and sometimes even a low abundance SV in a product can alter biological activity and trigger immune responses in the human body, leading to significant potential risks. Therefore, detecting such natural mutations before large-scale production is crucial. Our developed method can detect sequence differences as low as a single amino acid, and the detection range can cover the entire protein/antibody sequence.

Chinese/English project report

In the technical report, Biotech-Park will provide you with a detailed bilingual technical report in Chinese and English, including:

1. Experimental procedures (in Chinese and English)
2. Relevant mass spectrometry parameters (in Chinese and English)
3. Mass spectrometry images
4. Raw data
5. Peptide sequence information
6. Protein sequence and mutation information