Key Techniques and Application Advances in De Novo Protein Sequencing
De novo protein sequencing, as a core technology for determining the amino acid sequences of unknown proteins, has achieved significant breakthroughs in both methodology and application scenarios in recent years. Its primary advantage is that it does not rely on genomic or protein databases, enabling sequence reconstruction directly from mass spectrometry data. This approach is particularly useful for the study of proteins from newly discovered species, antibody therapeutics, and complex systems involving post-translational modifications (PTMs). This review provides an overview of recent advancements in this field, focusing on key technological innovations and the expansion of applications.
Key Techniques Breakthroughs
1. Advances in High-Precision Mass Spectrometry
Modern mass spectrometers, such as the Orbitrap Exploris and TimS TOF, achieve a mass accuracy of up to 0.001 Da, enabling the differentiation of isomeric amino acids like leucine and isoleucine. Coupled with novel fragmentation techniques, such as electron activation dissociation (EAD), the coverage and quality of fragment ion data have been greatly improved, thus establishing a solid foundation for the sequencing of long peptides.
2. Significant Progress in Intelligent Algorithms
(1) Deep Learning Models: Tools such as DeepNovo and PointNovo, trained on millions of mass spectrometry spectra, can automatically identify fragment ion patterns and predict sequences with an accuracy exceeding 90%.
(2) Prediction of Modification Sites: Algorithms can now simultaneously analyze post-translational modifications, including phosphorylation and glycosylation, thus reducing the cost of manual verification.
3. Standardization of Sample Processing
(1) Trace Sample Technology: Nano-scale liquid chromatography (nanoLC) combined with low-adsorption materials enables the efficient detection of nanogram-level proteins.
(2) Optimization of In Situ Enzyme Digestion: Gel-based enzyme digestion and solid-phase support technologies minimize sample loss and enhance sequencing success rates, particularly in complex mixtures.
Latest Application Advances in De Novo Protein Sequencing
1. Antibody Drug Development
De novo protein sequencing allows for the direct analysis of the variable region sequences of naturally occurring antibodies in patients, which significantly accelerates the design of personalized antibody-based therapeutics. For instance, in the rapid screening of neutralizing antibodies for COVID-19, this technology has played a pivotal role in identifying critical antigen epitopes.
2. Synthetic Biology and Artificial Proteins
In the field of synthetic biology, particularly with the design of artificial proteins such as enzymes and biomaterials, de novo protein sequencing is instrumental in confirming the sequence integrity of synthetic products, while also guiding the rational design of novel proteins.
3. Disease Biomarker Discovery
In the study of diseases like cancer and neurodegenerative disorders, de novo protein sequencing can identify low-abundance, post-translationally modified proteins that are often missed by conventional approaches. For example, it has facilitated the discovery of specifically phosphorylated Tau protein in the cerebrospinal fluid of Alzheimer's disease patients, which is crucial for diagnostic purposes.
4. Research on Biological Systems in Extreme Environments
Protein analysis of novel species, such as microorganisms from deep-sea hydrothermal vents or polar environments, is greatly enhanced by de novo protein sequencing. This supports the identification of novel biocatalysts, including heat-resistant DNA polymerases, which have significant applications in biotechnology.
De novo protein sequencing is evolving from a tool for “technical validation” to one for “broad-scale applications.” With continuous advancements in high-resolution mass spectrometry, innovative cleavage technologies, and improved computational tools, this method has demonstrated considerable advantages in areas such as identifying unknown sequences, detecting mutations, and analyzing post-translational modifications. These advancements are driving deeper applications in fields like antibody drug development, disease diagnostics, and protein function research. MtoZ Biolabs offers comprehensive de novo protein sequencing services. Our "one-stop" service streamlines the process, saving you time and effort, and enabling more efficient progress in your research.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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