De Novo Protein Sequencing

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The primary structure of the protein molecule itself is the core of its biological function. Therefore, accurate and rapid analysis of the complete sequence of the protein is conducive to the functional study of unknown proteins, and is of great significance for the development and production of protein drugs including monoclonal antibodies. De novo protein sequencing is a novel sequencing technology, it can directly determine the amino acid sequence of a protein without any known sequence or protein database information.

We Can Provide but Not Limited to:

  • Accurate determination of unknown protein or peptide sequence information
  • Accurate measurement after commercialization reform of protein and enzyme sequence
  • Accurate determination of stable cell line expressing protein sequence

Technology Platform of De Novo Protein Sequencing Service

De novo protein sequencing is based on the regular fragmentation of peptide molecules cleaved by protease in mass spectrometry, and finds specific fragmentation patterns, and then calculates the corresponding amino acid information and amino acid post-translational modifications based on the mass difference of the mass spectra.

Creative Proteomics uses de novo protein sequencing technology to perform the full length of protein sequence, mass spectrometry analysis of protein sequence, and positive and negative verification of protein sequence, so as to effectively and accurately analyze the full length of protein sequence.

De Novo Protein Sequencing Service

Advantages of De Novo Protein Sequencing Service

  • Rich experience in protein sequencing analysis
  • High accuracy and accuracy: The high resolution and sensitivity of the Orbitrap Fusion Lumos enhanced the reliability and accuracy of the results
  • Rigorous sequence prediction and rigorous sequence validation
  • Advanced data processing algorithm
  • Rapid turnaround time: 5-7 days to provide detailed technical reports
  • Customized service: We can customize professional solutions for you according to your research plan needs. You can select or suggest the required items for analysis

Creative Proteomics' analytical scientists can provide the sequence information of proteins or peptides related to the experiment, mass spectrometry image, original data and the full length sequence of proteins. Provide concise written reports to assist customers in de novo protein sequencing analysis of technical issues.


  1. Li Zhang, Yan Rent, et al. Rapid evolution of protein diversity by de novo origination in Oryza. Nature Ecology & Evolution, 2019, 3: 679-690.

De novo Protein Sequencing FAQ

In recent years, the emergent technology of de novo protein sequencing has piqued substantial scientific interest. This novel approach represents a unique analytical tool, enabling the deciphering of amino acid sequences without reliance on pre-existing sequence or protein database information. Its application holds particular pertinence to sequence discovery within the spheres of medical diagnostics and therapeutics. However, the complex sample requirements and procedural intricacies inherent in this method have been sources of critical concern. To ameliorate these reservations, we propose a detailed response to a host of frequently encountered queries as presented below.

Q: What is the difference between protein sequencing and gene sequencing?

A: The fundamental distinction between protein sequencing and gene sequencing lies in their respective levels of examination. Protein sequencing is primarily concerned with the amino acid level, while gene sequencing focuses on the nucleotide level. In situations where the nucleotide sequence is unknown or no reference database is available, protein sequencing can independently provide the complete amino acid sequence of the protein, thereby offering potent support for related research.

Q: What distinguishes protein de novo sequencing from the Edman degradation method of sequencing?

A: De novo sequencing employs algorithms for protein sequencing, directly inferring peptide sequences from ion information in mass spectra. Conversely, the Edman degradation method is typically suitable for peptides that consist of 15-20 amino acids and requires a high purity level for the sample, usually around 97% or above. Moreover, this method necessitates protein digestion, peptide purification, and individual, sequential machine tests. Therefore, the time and cost investments for monoclonal antibody sequencing through Edman degradation are comparatively substantial.

Contrasted with the traditional Edman method, mass spectrometry-based de novo sequencing displays superior efficiency, throughput, and cost-effectiveness. Given the known amino acid sequence of a protein, de novo sequencing can reveal new protein variants, which could emanate from unknown mutations, splice junctions, and various post-translational modifications, thus providing a more comprehensive picture of monoclonal antibody sequences.

Q: What types of samples are applicable to protein de novo sequencing?

A: Protein de novo sequencing is irrespective of the size or length of a protein, accommodating a wide range of sample types. This includes, but is not limited to, monoclonal antibodies (mAbs), Fab/Fc fragments, bispecific antibodies (bsAbs), multispecific antibodies, recombinant proteins, peptides, fluorescence-labeled antibodies, and cross-linked antibodies. Essentially, the technique is extraordinarily versatile, applicable to virtually all types of protein samples irrespective of their size and complexity.

Q: Can antibodies labeled with fluorescent markers or cross-linked to beads be used for de novo sequencing?

A: Indeed, both antibodies cross-linked to beads and those labeled with fluorescent markers such as FITC, Cy5, and PE are compatible with de novo sequencing processes, demonstrating the versatility of this approach.

Q: What are the sample requirements for protein de novo sequencing?

A: The standard requirement for protein de novo sequencing is a sample quantity of 100µg with a purity level exceeding 90% coupled with a proven track record of successful sequencing with at least 20µg. It is important to emphasize that protein purity considerably influences the outcome of the sequencing process. If the sample contains impurities such as antibody proteins, homologous proteins, or Bovine Serum Albumin (BSA), these could interfere with the sequencing process, thereby potentially compromising the experimental outcome.

Q: What is the required sample volume for antibody sequencing? Can the sequence be measured from mixed antibodies in serum without purification?

A: In antibody sequencing, the required sample volume varies depending on the sequencing type. For monoclonal antibody sequencing, as long as the purity of the antibody meets the requirement, 100 μg of antibody protein would suffice for sequencing. However, the scenario for sequencing mixed antibodies in serum is more complicated and requires a specific pre-initial assessment, to ascertain the appropriate sample volume.

Q: Does protein sequencing only provide the heavy chain sequence? Is it restricted to detecting only variable region sequences?

A: Our sequencing service employs comprehensive de novo sequencing to deliver a complete amino acid sequence report. This includes not only heavy and light chains, but also encompasses both the variable and constant regions.

Q: Does the composition of the sample buffer impact sequencing results?

A: For protein solubilization, our recommended buffer solutions typically involve PBS or Tris buffer systems. If the sample contains elements such as glycerol, BSA, detergent, or salt, we'll undertake appropriate purification techniques to mitigate their influence, thereby ensuring they do not adversely affect the sequencing output.

Q: Do glycosylation and post-translational modifications impact sequencing?

A: During the data analysis phase, potential post-translational and glycosylation modifications are taken into account. Therefore, such modifications do not affect the sequencing results of the sample itself.

Q: Can protein sequencing analyze post-translational modifications?

A: Utilizing advanced data analysis software, we are capable of identifying a range of common types of post-translational modifications, further pinpointing the corresponding modification sites. This provides substantial support for studying the functionality of proteins and their regulatory mechanisms within biological organisms.

Q: What kind of equipment is used in de novo protein sequencing?

A: The main equipment utilized in de novo protein sequencing comprises Thermo Fisher's tribrid ultra-high resolution mass spectrometers, specifically the Orbitrap Fusion and Orbitrap Eclipse models. With the continuous advancement in mass spectrometry technology, Creative Proteomics platform is actively committed to introducing more high-end devices to meet the diverse requirements of our clientele, thereby ensuring the delivery of premium quality service.

Q: What contents are included in the report of de novo protein sequencing?

A: The de novo protein sequencing report comprehensively features the following elements: (1) the full depiction of the protein amino acid sequence, inclusive of antibody heavy and light chain sequences, as well as constant and variable regions; (2) comprehensive molecular weight validation data, achieved through the comparison of observed molecular weight with theoretical sequence molecular weight, enabling the confirmation of sequence accuracy; (3) the portrayal of peptide coverage maps of an entire sequence, ensuring amino acid residue support from more than ten distinct peptide variants at each position; (4) the demonstration of secondary mass spectrograms supporting antibody variable region peptides; (5) the provision of a credibility analysis for I/L identification.

Q: What level of accuracy can be expected from de novo protein sequencing results?

A: To date, we have successfully completed thousands of projects. The accuracy of an individual amino acid sequence can reach 100%, with each amino acid in the sequence report being corroborated by as many as 30 or more different overlapping peptide spectra.

Q: Can protein sequencing delineate whether the identified peptides originate from human or microbial sources?

A: Absolutely, protein sequencing can pinpoint if the peptides being identified derive from humans or microbes. This discrimination is achieved through the analysis of species-specific unique peptides.

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