Advantages and Applications of De Novo Sequencing
-
Used for the structural elucidation of proteins with unknown or uncharacterized functions
-
Supports sequence comparisons among non-model species in evolutionary biology
-
Facilitates signaling pathway reconstruction and functional annotation, with particular value in species-specific protein analysis
-
Enables identification of mutation-derived neo-peptides, providing biomarker candidates for personalized diagnostics and therapy
-
Reveals disease-associated changes in protein modification states, such as tumor-specific phosphorylation profiles
-
Facilitates the discovery of novel antigens, contributing to the design of individualized immunotherapies
-
Applied to sequencing of hybridoma-derived or naturally occurring antibodies, supporting structure refinement and patent circumvention
-
Provides accurate sequence templates for antibody humanization and affinity maturation
-
Assists in domain identification and validation for bispecific and multispecific antibody formats
-
Used for sequence verification of synthetic peptides and recombinant proteins
-
Evaluates minor variations and mutation risks during manufacturing processes, strengthening quality control frameworks
-
Extensively employed in biosimilar comparability assessments, especially in structural consistency evaluation
-
Characterizes unannotated proteins from microbial communities, aiding the discovery of novel enzymes and metabolic pathways
-
Monitors biomarker proteins or stress-responsive proteins in environmental samples
-
Contributes to understanding the molecular adaptation mechanisms of extremophilic organisms
In proteomics research, the majority of mass spectrometry data analysis relies on database search approaches, which match experimental spectra with known protein sequences. De novo sequencing, a methodology that does not depend on any reference databases, has emerged as a powerful tool for elucidating previously unknown protein structures and functions.
Core Advantages of De Novo Sequencing
1. Fully Independent of Reference Databases
De novo sequencing derives peptide amino acid sequences directly from tandem mass spectrometry (MS/MS) data without referencing any external sequence databases. This provides distinct advantages for analyzing samples with limited or no annotation, such as non-model organisms, environmental samples, and ancient biological materials.
2. Capable of Identifying Novel Peptides Absent from Current Databases
In biological contexts involving cancer, infection, or genetic mutations, protein expression profiles often deviate significantly from standard reference datasets. De novo sequencing enables the discovery of novel peptides, alternative splicing products, fusion proteins, and other structural variants, laying the groundwork for mechanistic studies and therapeutic target identification.
3. Highly Sensitive to Mutations and Post-Translational Modifications
This technique can detect nonsynonymous mutations, amino acid substitutions, and small insertions or deletions (indels). It also facilitates comprehensive analysis of a wide range of post-translational modifications (PTMs), including phosphorylation, acetylation, and glycosylation.
4. Integration of Advanced Algorithms to Improve Sequencing Accuracy
Contemporary de novo sequencing workflows leverage a combination of computational strategies, such as tag-based spectral matching and graph-based algorithms, alongside machine learning-assisted interpretation. These integrations significantly enhance the precision and efficiency of peptide sequence reconstruction under complex biological conditions.
Major Application Areas of De Novo Sequencing
De novo sequencing finds broad applications in basic life sciences, clinical research, and industrial biotechnology.
1. Fundamental Research in Life Sciences
2. Studies on Disease Mechanisms and Precision Medicine
3. Antibody Discovery and Structural Verification
4. Industrial Biotechnology and Protein Analytics
5. Microbiome and Environmental Proteomics
Potential Challenges of De Novo Sequencing
Although de novo sequencing has demonstrated substantial capabilities across various domains, it still encounters several technical challenges:
1. Stringent Spectrum Quality Requirements
De novo sequencing is highly dependent on MS/MS spectra with high resolution and high signal-to-noise ratios. The quality of mass spectrometry data directly impacts the accuracy and comprehensiveness of peptide sequence interpretation, particularly in detecting low-abundance peptides.
2. Challenges in Interpreting Modifications and Isomers
Certain modification sites, such as isomeric phosphorylation and glycosylation, share identical mass characteristics, making them difficult to distinguish. When multiple modifications are present simultaneously, the resulting peptide fragmentation spectra become increasingly complex, highlighting the need for enhanced algorithmic performance as a key area for future development.
3. Absence of Standardized Validation Methods
Since de novo sequencing does not rely on reference databases, its results cannot be directly validated through database comparison. Instead, it requires complementary approaches such as transcriptome correlation, isotope labeling, or synthetic peptide verification, which collectively contribute to a more complex validation workflow.
De novo sequencing is not merely an analytical technique, but also serves as a strategic pathway to transcend database limitations and explore unknown protein structures and functions. As proteomics research continues to advance toward personalization, diversification, and non-standardization, the significance of de novo sequencing will become increasingly pronounced. MtoZ Biolabs is actively expanding its capabilities in mass spectrometry-based proteomics, from platform development to algorithm refinement, to provide high-quality, high-confidence de novo sequencing services that empower both academic and industrial research teams in their pursuit of deeper insights into the biological landscape.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
Related Services
How to order?
