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Protein De Novo Sequencing from MtoZ Biolabs

publication date: Aug 21, 2019
 | 
author/source: MtoZ Biolabs

Protein molecules play a key role in kit development, diagnostic reagent development, and drug discovery. The primary structure of the protein molecule itself is the core of its biological function. Therefore, the accurate and rapid analysis of the complete sequence of the protein contributes to the functional study of the unknown protein, and is significant to the development and production of protein drugs including monoclonal antibodies.

Overview-on-Protein-De-Novo-SequencingTraditional protein sequence analysis technology, whether based on MALDI-TOF mass spectrometer or nano LC-MS/MS platform, requires the use of a sequence database containing the identified proteins in the protein identification process, and then the molecular weight data measured by mass spectrometry can be compared to the molecular weight data obtained after fragmentation of the database theoretical sequence, so as to achieve sequencing and identification of the protein.

There are many proteins in the actual protein sequence analysis that are not included in existing databases, such as a new protein that has not been reported, or a protein Based on the above requirements for unknown protein sequence and mutation analysis, MtoZ Biolabs’ high-resolution mass spectrometer Obitrap Fusion Lumos combines rich bioinformatics analysis experience to establish a new generation of protein de novo sequencing and mutation analysis platform, enabling fast and accurate analysis of protein primary structures.

that were purified from a new species. In many cases, we can get theoretical data, but the missing part is the key. The commercially modified enzymes in the market, for instance. The sequence modification information of the enzyme is a technical barrier for competitors who intend to reverse engineer, because sequence changes are diverse (point mutations, deletions, insertions, substitutions, modifications, fusions, etc.). There are thousands of possibilities, and usually no directional cues can be found. Not only an enzyme in reagents box, many protein drugs also need to be optimized by mutation to increase yield or stability. In many cases, the protein sequence produces some mutations, which may cause changes in biological activity and produce an immune response even if there are few sequence mutations in a drug, and this in turn causes huge commercial losses. 

 

Protein De Novo Sequencing and Mutation Analysis Process

 

Platform Advantages 

  • Full sequence coverage of the protein (100% Sequence Coverage). In the process of protein sequencing, it will select 5 different proteases for enzyme digestion, and uses 100% full sequence splicing of protein molecules by complementarity between different peptide fragments.
  • Advanced data processing algorithms. Based on the literature published in Nature Biotechnology and the professional proteomics journal JPR, MtoZ Biolabs developed its own mass spectrometry raw data calculation method based on the existing software PEAKS Mass Spectrometry Software, which is specialized in database-independent protein sequence analysis. The principle of widening and narrowing out achieves accurate identification of protein sequences without missing any valid data information.
  • High precision and accuracy. It uses high-resolution and high-sensitivity Orbitrap Fusion Lumos mass spectrometers during sequencing. Compared to older generation mass spectrometers (such as Obitrap Elite, Q Exactive, etc.), Lumos improves the scanning speed of primary and secondary mass spectrometry. At the same time, the accuracy of the molecular weight of the peptide is ensured. The improvement of the scanning speed of the first-order mass spectrometer can help the same peptide segment to be identified more times, and the reliability of the result is improved. The scanning speed of the secondary mass spectrometer can be improved. Good fragmentation (as shown in the figure below) ensures the accuracy of the results, and accurate determination of leucine and isoleucine can be achieved by the characteristic peaks of the secondary fragment.
  • Strict sequence prediction and strict sequence verification. MtoZ Biolabs will first predict the sequence of the sequence, and will also create a new database with the sequence of the assay. It will analyze the collected mass spectrometry data again using the Sequence Confirmation mode. Only the exact match will be provided to customers.

 

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