What Is De Novo Sequencing?
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Multi-enzyme digestion combined with high-resolution mass spectrometry (Orbitrap Fusion Lumos)
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Proprietary De Novo assembly algorithms supplemented by manual curation
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Support for PTM identification, CDR region coverage, sequence modeling, and expression validation
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Compatibility with diverse protein classes, including antibodies, fusion proteins, recombinant enzymes, and mutant proteins
De Novo sequencing is a technique that reconstructs the original primary structure of a biomolecule—such as DNA, RNA, or proteins—directly from experimental data, without relying on any prior sequence information or reference databases. It serves as a critical approach for investigating novel species, new mutants, unknown antibodies, natural proteins, and other unexplored biological samples. In proteomics, De Novo sequencing involves the use of high-resolution tandem mass spectrometry (MS/MS) to analyze fragment ions of enzymatically digested peptides. Specialized software algorithms interpret the resulting ion spectra to deduce amino acid residues sequentially, ultimately assembling the full-length protein sequence. In contrast to traditional database-dependent methods, De Novo sequencing represents a true structure-based inference strategy, enabling the discovery of previously unannotated proteins, mutant variants, and post-translational modifications that are absent from existing databases.
Comparison Between De Novo Sequencing and Database-Dependent Sequencing
Applications of De Novo Sequencing
De Novo sequencing is no longer confined to exploratory studies in cutting-edge laboratories; it is now a critical and increasingly indispensable tool in antibody drug development, non-model organism research, bioinformatic discovery, and biopharmaceutical quality control.
1. Antibody Structure Reconstruction and Expression Template Design
Antibodies constitute one of the most important classes of therapeutic proteins. However, in practical scenarios, researchers and biopharmaceutical developers often possess only the antibody protein sample, with no access to its original gene sequence or cellular source. In such cases, De Novo sequencing enables direct enzymatic digestion and mass spectrometric analysis of the antibody protein, reconstructing the complete amino acid sequences of both heavy and light chains—particularly the complementarity-determining regions (CDRs), which are critical for antigen binding. These sequences can then be used for cDNA reverse engineering, expression system design, and functional validation. This technology facilitates the reproduction of legacy antibodies, development of biosimilars, and rational design of affinity-enhancing mutations, thereby significantly improving the efficiency of antibody engineering.
2. Analysis of Vaccine-Induced Antibody Repertoires and Clinical Immunology
In the context of vaccine development and immune profiling, there is growing interest in evaluating whether vaccine-induced polyclonal antibodies exhibit high affinity and potent neutralizing capacity. Traditional B-cell receptor (BCR) sequencing is unable to reflect the actual post-translational modifications or conformational states of antibodies. In contrast, De Novo sequencing directly analyzes antibody proteins present in serum or tissue fluids, capturing their authentic expression forms and modification patterns. This allows for detailed reconstruction and functional enrichment of antibody clonal repertoires. The technique has become especially valuable in fields such as cancer immunotherapy, HIV research, and neutralizing antibody development, where it serves as a powerful tool for elucidating complex immune responses.
3. Protein Analysis in Non-Model Organisms and Unknown Species
In the study of non-model organisms such as plants, microorganisms, marine species, and bioactive components of traditional medicine, proteomic databases are often incomplete or lack sufficient annotation. De Novo sequencing, which does not rely on reference genomes or known protein sequences, is well-suited for investigating protein expression in newly identified species, novel tissues, or previously uncharacterized biological states. For instance, in research on protein-based bioactive compounds within traditional medicine, De Novo sequencing can directly identify active proteins from complex biological extracts, thereby advancing the development of natural therapeutics.
4. Consistency Assessment and Quality Control in Biopharmaceutical Production
During biopharmaceutical manufacturing, subtle structural variations may arise between production batches or across different expression systems (e.g., CHO vs. HEK293 cells), potentially altering post-translational modification (PTM) profiles. De Novo sequencing enables comprehensive reconstruction and comparison of the full-length expressed protein structures, allowing detection of not only prominent mutations but also nuanced modifications such as glycosylation, oxidation, and structural isomerization. This capability supports rigorous consistency evaluation, facilitates the development of biosimilars, and enhances the preparation of regulatory submissions.
5. Identification of Cancer Neoantigens and Personalized Immunotherapy
Cancer cells frequently generate unique mutated peptide fragments, termed neoantigens, which serve as central targets in cancer vaccines and T-cell-based immunotherapies. Conventional proteomics approaches often fail to detect these low-abundance peptides due to sequence alterations or post-translational modifications. De Novo sequencing, however, can identify previously unannotated peptides that are uniquely expressed in tumor tissues. When integrated with immunogenicity prediction tools, this approach enables precise targeting for individualized cancer therapies.
6. Mapping Protein Modifications and Investigating Structure–Function Relationships
The biological activity of many proteins is modulated by post-translational modifications (PTMs), including glycosylation, acetylation, and phosphorylation. De Novo sequencing can accurately reconstruct the amino acid sequence of the protein backbone while simultaneously identifying the types, sites, and occupancy levels of PTMs from high-resolution mass spectrometry data. This provides critical insights into protein functional states, conformational dynamics, and interaction mechanisms, making it a powerful tool in fields such as structural biology and signal transduction research.
De Novo Sequencing Services at MtoZ Biolabs
MtoZ Biolabs offers high-accuracy, high-coverage, and highly reproducible De Novo sequencing solutions tailored for antibody characterization, protein drug development, and disease mechanism studies. Our technical strengths include:
Whether you are a researcher or a biopharmaceutical developer, you can simply provide your antibody or protein sample, and we will deliver complete structural information—suitable for recombinant expression and patent design—within 3 to 4 weeks. In an era increasingly dependent on existing databases, De Novo sequencing offers a complementary capability: decoding proteins from scratch, independent of prior annotations. It is not a replacement for database-driven approaches, but rather the most reliable, flexible, and exploratory strategy for addressing novel challenges, complex samples, or unknown proteins. At MtoZ Biolabs, we are committed to empowering our clients to move beyond database constraints and uncover the full complexity of protein structures through state-of-the-art experimental and computational platforms.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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