Common Issues in De Novo Sequencing and How to Solve Them
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Decreased digestion reproducibility and reduced sequence coverage
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Potential loss of information related to low-abundance modifications or isomeric variants
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Full-length antibody sequence reconstruction from as little as 10 µg of starting material
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Micro-scale enrichment strategies for challenging samples (e.g., IP-derived antibodies)
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A “multi-pass digestion + integrative data interpretation” strategy to ensure reliability
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Sample complexity (purity, presence of post-translational modifications)
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Requirements for auxiliary analyses such as PTM mapping or structural modeling
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Inclusion of recombinant expression and functional validation steps
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Sequence reconstruction of clinical polyclonal antibodies
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Profiling of B-cell repertoires from vaccine-immunized mice
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Patent antibody reconstitution and structure-based validation
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Full-length mapping of post-translationally modified antibodies (e.g., glycosylated variants)
In proteomics and antibody-related studies, De Novo Sequencing has emerged as a powerful approach due to its independence from reference databases and its ability to reconstruct complete protein sequences. This capability makes it indispensable for applications such as antibody sequence characterization, ancient protein reconstruction, and novel antigen discovery—contexts where De Novo Sequencing is often the only viable solution. However, owing to its reliance on complex mass spectrometry data interpretation and computational reconstruction algorithms, researchers frequently encounter various uncertainties at the initial stages of a project. This article summarizes the seven most commonly asked questions during project consultations and offers expert insights and recommendations.
Q1: Can De Novo Sequencing guarantee “100% accuracy”?
Answer: Theoretically, no; practically, it can achieve very high accuracy.
De Novo Sequencing depends on MS/MS fragmentation spectra of peptides, and its accuracy is influenced by:
(1) Enzymatic digestion coverage
(2) Quality of MS/MS fragment ions
(3) Ability to resolve isobaric or isomeric peptides
Under optimized conditions—such as the use of multiple proteases, high-resolution mass spectrometers, and manual sequence validation—the sequence recovery rate can exceed 95%. Critical regions, especially the Complementarity-Determining Regions (CDRs), are typically subjected to enhanced coverage and verification. At MtoZ Biolabs, a comprehensive workflow integrating dual-platform acquisition, manual review, and structure-based modeling ensures that the final sequences support recombinant expression and functional reconstitution.
Q2: Is it necessary to use purified protein samples? Can sequencing be performed directly from serum or cell supernatant?
Answer: Purification is not strictly required, but strongly recommended.
Reliable De Novo Sequencing requires efficient proteolytic digestion and high-quality fragment ion generation. Direct analysis from complex biological matrices can result in significant peptide interference, especially since antibody light and heavy chains are often present at much lower levels compared to abundant plasma proteins (e.g., albumin, other immunoglobulins).
To address this, MtoZ Biolabs offers:
(1) Protein A/G affinity purification
(2) Pre-purification using SDS-PAGE or SEC
(3) Antigen-specific antibody pull-down enrichment
These steps ensure the acquisition of high-purity samples amenable to De Novo Sequencing, even from challenging sources such as serum, tissue fluid, or low-expression cell lines.
Q3: Is a high-resolution mass spectrometer alone sufficient for De Novo Sequencing?
Answer: No—instrumentation, computational algorithms, and expert interpretation are all critical.
While advanced instruments like Orbitrap and TOF provide high-accuracy fragmentation data, the core challenges of De Novo Sequencing include:
(1) Designing optimal multi-protease digestion strategies
(2) Correctly assembling overlapping peptide fragments
(3) Resolving ambiguities among near-identical sequences
(4) Post-processing and expert-guided sequence curation
At MtoZ Biolabs, proprietary algorithms are integrated with leading software tools (e.g., PEAKS, pNovo, Novor), supported by manual validation and structure-informed analysis, to enable seamless translation from raw MS data to an expressible sequence.
Q4: Can De Novo Sequencing detect protein post-translational modifications (PTMs)?
Answer: Yes, provided that the experimental workflow is tailored accordingly.
Typical PTMs such as glycosylation (N-linked and O-linked), phosphorylation, methylation, acetylation, oxidation, and deamidation can significantly alter fragmentation patterns and challenge de novo interpretation algorithms. To improve PTM detection accuracy, the following steps are essential:
(1) Pre-specification of the modification types of interest
(2) Use of appropriate enrichment strategies (e.g., HILIC, IMAC)
(3) Activation of targeted PTM search parameters during analysis
Q5: Given the critical role of CDRs, can 100% sequence recovery be guaranteed?
Answer: The accuracy of CDR reconstruction is a key evaluation criterion in De Novo sequencing.
The complementarity-determining regions (CDRs) of antibodies are responsible for antigen binding and exhibit high sequence variability. Due to the lack of matching entries in public databases, their identification relies entirely on De Novo reconstruction. To ensure accurate CDR recovery, we typically employ:
(1) Complementary protease digestion using combinations such as Trypsin, Chymotrypsin, and GluC
(2) Redundant enzymatic digestion to enhance sequence coverage
(3) CDR-targeted enrichment algorithms
(4) Cross-validation with antibody structural templates
These strategies collectively enable high-confidence identification and reconstruction of CDRs, achieving >95% sequence coverage and full functional recovery in most cases.
Q6: Is De Novo sequencing feasible with only 10 µg of protein?
Answer: Yes, although we recommend using >20 µg to ensure optimal data quality.
Contemporary high-sensitivity mass spectrometers allow MS/MS acquisition under low-input conditions. However, reduced sample amounts can lead to:
MtoZ Biolabs has developed a dedicated workflow optimized for low-input De Novo sequencing, supporting:
Q7: What is the typical turnaround time? When can I expect the complete antibody sequence?
Answer: Standard turnaround is 3–4 weeks; expedited processing is available within 10 business days.
The total project duration may vary depending on:
MtoZ Biolabs offers flexible service tiers to accommodate project timelines. Our technical team provides end-to-end support, from sample reception and experimental design to data analysis and expression validation, ensuring the fastest delivery of reproducible and expression-ready antibody sequences.
MtoZ Biolabs: Your One-Stop Platform from De Novo Sequencing to Functional Validation
As a mass spectrometry-driven antibody research provider, MtoZ Biolabs has completed over 200 complex De Novo antibody sequencing projects, including:
We adopt a rigorous, three-pronged strategy—combining algorithmic precision, expert interpretation, and experimental validation—to tackle complex sequencing challenges. Given its technical demands and interpretive complexity, De Novo sequencing requires a sound understanding of its principles and limitations prior to project initiation. We hope this FAQ provides clarity for your project planning and scientific decision-making. For inquiries related to sample preparation, experimental design, or technical details, please reach out to our consultants or submit your questions through our official website. We are committed to delivering tailored, expert support as promptly as possible.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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