Protein Sequencing Technology
Protein sequencing technology is fundamental to determining the amino acid sequences of proteins, providing critical insights into their structure-function relationships. This technology is widely applied in medicine, drug discovery, and biotechnology. In disease research, protein sequencing aids in identifying disease-associated protein variants, supporting early diagnosis and personalized treatment. Furthermore, sequencing specific proteins enables scientists to design engineered proteins with tailored functions, accelerating advancements in bioengineering.
The development of protein sequencing technology began in the 1950s with the Sanger method. Over time, scientific and technological innovations have driven rapid advancements in sequencing techniques. Modern approaches now support both individual protein sequencing and high-throughput sequencing of complex protein mixtures, significantly improving research efficiency and accuracy.
Traditional Protein Sequencing Techniques
1. Edman Degradation
The Edman degradation method selectively reacts with the N-terminal amino acid using phenyl isothiocyanate (PITC) under weak alkaline conditions, forming a phenylthiocarbamyl (PTC)-protein derivative. The N-terminal amino acid is then cleaved as a phenylthiohydantoin (PTH)-amino acid under acidic conditions, allowing sequential identification of the protein's amino acid sequence.
(1) Advantages: This method offers precise N-terminal amino acid sequencing, particularly effective for small proteins or peptides. It is also capable of determining N-terminal sequences in protein mixtures.
(2) Disadvantages: It is limited by its inability to sequence chemically modified N-terminals, halts at non-α-amino acids, and has low efficiency for large proteins, typically not identifying disulfide bond positions.
2. Peptide Mapping
Peptide mapping involves enzymatic or chemical fragmentation of proteins, followed by mass spectrometry to generate peptide mass fingerprints. These fingerprints are then compared with theoretical maps in protein databases for protein identification and partial sequence information.
(1) Advantages: The peptide fragments are compatible with the scanning range of mass spectrometers, allowing for high coverage analysis when using specific and non-specific proteases.
(2) Disadvantages: Identification is limited to known database proteins, and contamination can affect accuracy since it only determines peptide mass.
Mass Spectrometry-Based Protein Sequencing Techniques
1. Peptide Mass Fingerprinting
In peptide mass fingerprinting, proteins are enzymatically digested, and the resulting peptide fragments are analyzed for their mass/charge ratio (m/z) using mass spectrometry. The experimental fingerprints are compared with theoretical ones in databases for protein identification.
(1) Advantages: The method is straightforward and rapid, suitable for simultaneous analysis of multiple proteins and large-scale identifications.
(2) Disadvantages: It heavily depends on existing database entries and may struggle with complex protein mixtures.
2. Tandem Mass Spectrometry (MS/MS)
Tandem mass spectrometry involves selecting specific peptide ions for fragmentation into smaller ions, which are then analyzed to deduce the peptide's amino acid sequence.
(1) Advantages: This technique provides detailed sequence information, supports de novo sequencing without database reliance, and excels in sensitivity and resolution for low-abundance proteins.
(12) Disadvantages: It requires pure samples and complex data analysis, with ion suppression effects in mixed samples potentially affecting accuracy.
Emerging Protein Sequencing Techniques
1. Nanopore Sequencing
Nanopore sequencing detects changes in ionic current as protein molecules pass through nanopores. Different amino acids generate distinct current signals, enabling sequence determination through signal analysis.
(1) Advantages: It allows single-molecule sequencing and direct detection of post-translational modifications without labeling, facilitating real-time interaction studies.
(2) Disadvantages: The technology is still maturing, with challenges in accuracy, throughput, and complex protein sequencing.
2. Machine Learning-Based Sequencing
This approach uses machine learning models trained on known protein sequences and associated data features to predict amino acid sequences of unknown proteins.
(1) Advantages: Capable of handling complex data, integrating diverse information for enhanced accuracy and efficiency, and analyzing large-scale proteomics data.
(2) Disadvantages: It requires extensive high-quality training data, and model accuracy depends on data diversity. The approach may struggle with novel proteins or those with unique structures and functions.
The Significance of Protein Sequencing Technology
Protein sequencing technology offers high-resolution sequence information crucial for understanding protein functions. High-throughput capabilities enable large-scale sample analysis, significantly advancing research efficiency. Moreover, protein sequencing plays an indispensable role in discovering novel proteins and elucidating complex biological systems. MtoZ Biolabs delivers comprehensive protein sequencing services within the proteomics domain. Our experienced team provides expert technical support and customized sequencing solutions, empowering clients to achieve breakthroughs in research and industrial applications. We invite researchers to collaborate with us in advancing life sciences.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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