Protein Sequencing: Methods, Technologies & Applications
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High accuracy
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Suitable for determining N-terminal sequences of individual proteins
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Inapplicable to complex mixtures
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Limited to N-terminal analysis; does not detect post-translational modifications
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Low throughput; unsuitable for large-scale proteomics studies
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Efficient and highly automated
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Relies on curated databases (e.g., UniProt, NR, Swiss-Prot)
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Compatible with peptide quantification techniques (e.g., TMT, iTRAQ)
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Bottom-up: Proteins are digested into peptides prior to MS analysis. This method is widely used due to its high sensitivity and throughput, especially for complex samples.
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Top-down: Intact proteins are analyzed directly, enabling detailed characterization of structural variants and post-translational modifications. However, it requires higher-resolution instrumentation and is typically applied to structural or variant proteins.
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Phosphorylation sites within kinase-regulated signaling networks
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The association between histone modifications (acetylation, methylation) and gene expression
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Structural and functional disruptions resulting from splicing variants
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Confirmation of antibody sequences (distinguishing heavy and light chains)
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Stability assessment (detection of microheterogeneity and point mutations)
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Impurity profiling (monitoring for immunogenic risk)
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Screening of agricultural germplasm resources
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Discovery of functional proteins in marine species
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Investigation of enzymatic activities related to natural products in insects and microbes
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Phosphorylation
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Acetylation
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Ubiquitination
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Glycosylation
Proteins are key functional components within cells, and their biological activity is determined by their precise amino acid sequences. Alterations in these sequences can significantly impact a protein’s structure, interactions, and function. Protein sequencing not only elucidates protein mechanisms but also plays a fundamental role in disease research, antibody development, and the identification of novel drug targets. Since the mid-20th century, sequencing technologies have evolved significantly—from the Edman degradation method to high-throughput mass spectrometry platforms—resulting in enhanced coverage and data precision. These advances have made protein sequencing a cornerstone of modern proteomics.
Main Methods for Protein Sequencing
1. Edman Degradation
Edman degradation is a chemical sequencing method that removes amino acids sequentially from the N-terminus of a peptide or purified protein. The method relies on the selective labeling of the N-terminal residue with phenyl isothiocyanate (PITC), followed by chromatographic identification of the cleaved derivative.
🔸 Technical Advantages
🔸 Main Limitations
Although its usage has declined with the rise of high-throughput mass spectrometry, Edman degradation remains valuable in specific applications such as validating N-terminal modifications or identifying recombinant proteins.
2. Mass Spectrometry-Based Sequencing
Mass spectrometry (MS) is now the predominant approach for protein sequencing. It involves enzymatic digestion of proteins followed by analysis of the resulting peptide fragments using high-resolution MS platforms to deduce the protein’s primary structure.
🔸 Common Strategies in MS-Based Sequencing
(1) Database-Dependent Sequencing
This approach identifies protein sequences by matching MS/MS spectra to theoretical peptide profiles from known protein databases.
▸Key Characteristics:
Common software tools: Mascot, Sequest, MaxQuant, Proteome Discoverer
(2) De novo Sequencing (Database-Independent)
In cases where reliable database references are lacking or when discovering novel mutations and peptide variants, de novo sequencing is preferred. It directly infers amino acid sequences from MS/MS spectra without database reliance. Popular tools include PEAKS, Novor, pNovo, and DeepNovo.
(3) Top-Down vs. Bottom-Up Strategies
Mass spectrometry-based sequencing is often categorized by the analytical approach:
Applications of Protein Sequencing
1. Biomarker Discovery
By comparing protein sequence variations across samples in different physiological or pathological states (e.g., healthy vs diseased, treatment vs control), researchers can identify potential biomarkers, including disease-associated mutant peptides, neoantigens, and alternative splicing variants.
2. Investigation of Disease Mechanisms
Alterations in protein sequences or post-translational modifications (PTMs) are central to the pathogenesis and progression of many diseases. Protein sequencing enables the identification of:
3. Antibody Sequencing and Biopharmaceutical Development
Protein sequencing plays a pivotal role in the development of antibody-based biopharmaceuticals. Major applications include:
4. Studies on Non-Model Organisms and Novel Species
For organisms lacking genomic references, de novo protein sequencing via mass spectrometry enables direct acquisition of essential protein sequence information. Representative applications involve:
5. PTM Site Mapping and Functional Analysis
Post-translational modifications (PTMs) are fundamental regulatory mechanisms governing protein activity. Common PTMs include:
Through targeted enrichment strategies coupled with mass spectrometry, researchers can localize PTM sites and quantify their abundance at the proteome scale, offering insights into complex regulatory networks.
Protein sequencing acts as a vital bridge between genetic information and protein functionality. A comprehensive understanding of its methodologies and applications is crucial. MtoZ Biolabs is dedicated to providing an end-to-end protein sequencing solution—from sample to data—leveraging cutting-edge instrumentation and an experienced mass spectrometry team to deliver high-quality, traceable sequencing services that drive forward scientific and industrial discoveries.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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