De Novo Peptide Sequencing via Tandem Mass Spectrometry
In proteomics research, database-dependent mass spectrometry data analysis (database search) has become the predominant approach. However, in emerging fields such as non-model organisms, natural products, antibody engineering, and novel peptide therapeutics, the absence or incompleteness of reference databases limits the applicability of traditional methods. In such cases, de novo peptide sequencing—a technique that reconstructs peptide amino acid sequences directly from mass spectrometry data without relying on existing databases—becomes indispensable. The success of de novo peptide sequencing fundamentally depends on the acquisition and interpretation of high-quality tandem mass spectrometry (MS/MS) data. This article discusses the technical principles and application scenarios of performing de novo peptide sequencing via tandem mass spectrometry.
What Is De Novo Peptide Sequencing and Why Use MS/MS?
De novo peptide sequencing refers to the inference of the amino acid composition and sequence of a peptide by analyzing its MS/MS fragmentation spectra in the absence of any database reference. This process relies on the mass differences between fragment ions, primarily b-ions and y-ions, observed in the spectra.
Achieving this goal requires high-resolution, high-sensitivity tandem mass spectrometry. By subjecting selected precursor peptide ions to fragmentation and analyzing the resulting product ions, highly specific fragment information is obtained—currently the most reliable experimental basis for de novo sequencing.
Principles and Workflow of Tandem Mass Spectrometry
1. Peptide Generation and Ionization
Protein samples are enzymatically digested (e.g., using trypsin or LysC) to generate short peptide fragments. These fragments are introduced into the mass spectrometer and ionized via electrospray ionization (ESI), producing multiply charged peptide ions.
2. Primary Mass Spectrometry (MS1) Selection
The MS1 scan measures the mass-to-charge ratios (m/z) of all ionized peptides. Selected target peptides are then isolated for further fragmentation.
3. Fragmentation and MS/MS Acquisition
Targeted peptides are fragmented using methods such as collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), or electron transfer dissociation (ETD). The resulting b- and y-ion series form the fragmentation spectrum, which serves as the foundational dataset for de novo peptide sequencing.
4. Spectrum Interpretation and Sequence Reconstruction
Computational algorithms analyze the fragmentation spectrum by calculating the mass differences between adjacent fragment ions. These differences correspond to individual amino acids, enabling the stepwise reconstruction of the complete peptide sequence.
Advantages of Tandem Mass Spectrometry in De Novo Peptide Sequencing
1. High Throughput: Tandem mass spectrometry allows for the analysis of thousands of peptides in a single analytical run, making it highly suitable for complex biological samples.
2. High Specificity: Fragmentation spectra provide structure-specific information, enabling the identification of peptides with previously unknown sequences.
3. Broad Compatibility with Modifications: This approach can reliably detect various post-translational modifications (PTMs), including phosphorylation, acetylation, and oxidation.
4. Sensitive Detection of Low-Abundance Peptides: Advanced high-resolution instruments such as Orbitrap and timsTOF Pro enable accurate sequencing of peptides present at low abundance.
Representative Applications: From Basic Research to Drug Discovery
1. Monoclonal Antibody Sequence Characterization
Many antibodies produced by hybridoma cells lack annotated sequences. De novo sequencing enables precise identification of complementarity-determining regions (CDRs) within both light and heavy chains, offering valuable guidance for antibody humanization and therapeutic design.
2. Discovery of Natural Bioactive Peptides
In studies involving antimicrobial peptides, neuropeptides, and plant defense peptides, de novo sequencing facilitates the identification of novel natural peptide candidates with potential pharmaceutical relevance.
3. Proteomic Analysis in Non-Model Organisms
For organisms such as plants, insects, and marine species that lack comprehensive protein databases, de novo sequencing provides the only viable approach for reconstructing protein sequences.
4. Identification of Novel Translational Products (sORFs)
By integrating de novo sequencing with transcriptomic data, previously unannotated short open reading frames (sORFs) can be revealed, opening avenues for the exploration of novel regulatory mechanisms.
Leveraging years of experience in proteomics platform development, MtoZ Biolabs has established a robust, high-throughput, and industry-ready de novo peptide sequencing workflow, offering the following key advantages:
(1) Cutting-Edge Instrumentation: Utilization of state-of-the-art platforms such as Orbitrap Eclipse and timsTOF Pro 2 ensures the generation of high-quality MS/MS data;
(2) Integrated Intelligent Algorithms: Incorporation of DeepNovo, PEAKS, and proprietary deep learning models enhances sequence identification accuracy;
(3) Flexible Enzyme and PTM Strategies: Supports customized multi-enzyme digestion workflows tailored for diverse sample complexities and modification types;
(4) Comprehensive Validation Support: Confirmatory analyses including synthetic peptide validation and functional assays ensure sequence accuracy and reliability.
De novo peptide sequencing—particularly approaches leveraging high-resolution tandem mass spectrometry—has emerged as a powerful tool for uncovering previously uncharacterized proteins and functional peptide segments. This method not only bridges the limitations of database-dependent searches but also exhibits significant promise in drug discovery, structural biology, and synthetic biology.
MtoZ Biolabs remains committed to advancing intelligent proteomics technologies, delivering precise and efficient de novo sequencing services to both academic researchers and biopharmaceutical enterprises. For professional technical consultation and customized solutions, we invite you to contact us at any time.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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