Amino Acid Sequence Analysis: Techniques and Challenges
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High-resolution mass spectrometry systems (Orbitrap Exploris, Fusion Lumos) enabling comprehensive sequencing and precise identification of intact protein peptides and their modifications
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Standardized sample preparation protocols (including reduction, alkylation, and automated enzymatic digestion) to ensure consistent sample quality
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Advanced multi-strategy data analysis pipelines (combining database matching with parallel de novo approaches) to enhance sequence reconstruction accuracy
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Capabilities for sequence alignment, mutation detection, PTM annotation, and data visualization
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Cross-platform integration to support a range of applications, including protein sequencing, functional genomics, and antibody validation
The amino acid sequence forms the foundation of protein function and serves as a crucial entry point for understanding regulatory mechanisms in organisms, disease progression, and the action of molecular drugs. With the rapid advances in proteomics, amino acid sequence analysis has evolved from traditional chemical determination methods to high-throughput, modifiable, and multidimensional data integration approaches, establishing itself as a vital link between genomics and phenotypic function. This paper discusses the current mainstream techniques for amino acid sequence analysis and the associated challenges.
Core Techniques of Amino Acid Sequence Analysis
1. Tandem Mass Spectrometry (LC-MS/MS)
The most widely used approach for amino acid sequence analysis today is mass spectrometry sequencing based on LC-MS/MS, which involves the following steps:
(1) Enzymatic digestion: The target protein is digested into peptide fragments using trypsin or other specific proteases.
(2) Liquid chromatography separation: Complex peptide mixtures are separated using high-performance liquid chromatography to improve resolution.
(3) Primary mass spectrometry (MS1): Detection of precursor ion mass-to-charge ratios (m/z) to preliminarily screen peptide fragments.
(4) Secondary mass spectrometry (MS2): Collision-induced dissociation generates b and y ion series, and amino acid sequences are deduced from mass differences between fragment ions.
(5) Database matching and sequence identification: Platforms such as MaxQuant, PEAKS, and Byonic are used to reconstruct peptide sequences and map them to protein databases.
2. De Novo Sequencing
For studies lacking database references or involving unknown proteins, de novo algorithms reconstruct amino acid sequences directly from fragmentation spectra, making this approach well-suited for research on new species or antibody variable region characterization.
3. Edman Degradation
This technique identifies the sequence of short peptides by stepwise chemical removal of N-terminal amino acids followed by analysis. Despite its high accuracy, it has largely been supplanted by mass spectrometry due to limitations in throughput and versatility.
Challenges in Amino Acid Sequence Analysis
1. Protein Complexity
Proteins can undergo alternative splicing and post-translational modifications (PTMs), resulting in the formation of multiple isoforms that considerably increase the complexity of sequence analysis.
2. Uncertainty of Modification Sites
PTMs, including phosphorylation, acetylation, and methylation, often induce mass shifts or alter fragmentation pathways, thereby complicating the accurate localization of modification sites.
3. Sample Processing Variability
Inadequate control over protein extraction, enzymatic digestion, and purification steps can lead to reduced sequencing efficiency, increased background noise, and potential interruptions in sequence reconstruction.
4. Data Analysis Pressure
Handling large volumes of fragment spectra necessitates efficient and precise database matching, as well as manual validation, both of which are critical factors determining the overall quality of sequence reconstruction.
MtoZ Biolabs' Amino Acid Sequence Analysis Platform
To address these challenges, MtoZ Biolabs has developed an integrated protein sequence analysis platform characterized by the following capabilities:
Amino acid sequence analysis is fundamental not only for understanding protein structure but also for elucidating functional states, disease mechanisms, and drug interactions. With ongoing advancements in mass spectrometry technologies and continuous improvement of analytical algorithms, accurate, high-throughput, and modification-specific amino acid sequence analysis is poised to become a standard approach in protein research. MtoZ Biolabs remains committed to providing state-of-the-art platforms and expert services to drive life science research and biotechnology innovation. For customized solutions in protein sequencing, antibody validation, and modification analysis, please contact us.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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