Understanding and Analyzing Protein Primary Structure
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It accommodates complex samples and high-molecular-weight proteins
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It enables simultaneous analysis of multiple proteins and modification sites
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Its integration with database searching yields broad sequence coverage and high analytical efficiency
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Dual-platform support (Edman degradation and mass spectrometry) for diverse sample types and analytical needs
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Standardized digestion and purification protocols to enhance result consistency
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State-of-the-art mass spectrometry systems (Orbitrap Exploris, Fusion Lumos) for high-accuracy sequencing
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Comprehensive data interpretation and reporting, with options for FASTA format export and mutation/modification annotations
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Integration with secondary structure prediction, functional annotation, and bioinformatics analysis
Introduction: The Starting Point of Protein Structure Research
The primary structure of a protein refers to the linear sequence of amino acids, which determines its higher-order spatial arrangements—including secondary, tertiary, and quaternary structures—and ultimately its biological functions. This sequence not only represents the expression of the genetic information encoded by the protein but also forms the foundation for its functional realization.
Accurately deciphering the primary structure of proteins is crucial for elucidating protein functional mechanisms, identifying mutation sites, and facilitating the development of therapeutic antibodies and vaccines. Currently, primary structure analysis relies primarily on two core techniques: Edman degradation and mass spectrometry.
Major Technical Principles of Primary Structure Analysis
1. Edman Degradation: A Classical Chemical Sequencing Method
Edman degradation enables precise sequencing of short peptides by sequentially removing and identifying amino acids from the N-terminus of a protein. While highly specific and yielding clear results, this technique has several limitations:
(1) Applicable only to peptides containing 20–50 amino acids
(2) Ineffective for proteins with blocked or chemically modified N-termini
(3) Low throughput, rendering it challenging for complex sample analysis
2. Mass Spectrometry Sequencing: A High-Throughput Mainstream Approach
Currently, the predominant method for protein primary structure analysis is Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS), which operates as follows:
(1) Proteins are enzymatically digested, commonly with trypsin, into peptide fragments
(2) The peptides are separated by liquid chromatography (LC) and introduced into the mass spectrometer
(3) MS1 detects the precursor ion mass, while MS2 conducts fragmentation analysis
(4) Software algorithms compare the resulting fragmentation spectra with database entries to deduce amino acid sequences
Mass spectrometry sequencing offers distinct advantages:
Standard Workflow for Analyzing Protein Primary Structure
Step 1: Protein Extraction and Quantification
Extract proteins from cells, tissues, or serum samples, and normalize the protein concentration to a standard level.
Step 2: Reduction, Alkylation, and Enzymatic Digestion
Treat samples with reducing agents (DTT) and alkylating agents (IAA), followed by trypsin digestion to generate peptides amenable to mass spectrometric analysis.
Step 3: LC-MS/MS Sequencing
Separate peptides by liquid chromatography and analyze them using tandem mass spectrometry (MS/MS), recording fragmentation patterns for sequence determination.
Step 4: Data Analysis and Sequence Identification
Employ software tools such as MaxQuant, PEAKS, and Byonic for sequence reconstruction, identification of mutation sites, and detection of post-translational modifications.
Applications of Protein Primary Structure Analysis
1. Identification of Novel Proteins and Functional Prediction
The primary structure provides a molecular foundation for newly identified proteins, supporting structure modeling and functional annotation.
2. Detection of Disease-Associated Mutations
Comparing the primary structure between pathological and control samples facilitates the identification of pathogenic variants, including point mutations and splicing alterations.
3. Quality Control of Biopharmaceutical Products
For biologics such as antibodies and recombinant proteins, sequencing the primary structure verifies correct expression and ensures purity consistency.
4. Localization of Post-translational Modifications (PTMs)
Mass spectrometry enables simultaneous identification of phosphorylation, acetylation, and other modification sites, aiding in the elucidation of protein regulatory mechanisms.
MtoZ Biolabs Protein Primary Structure Analysis Services
MtoZ Biolabs offers comprehensive primary structure analysis services for research and biopharmaceutical applications, including:
The primary structure of a protein is the fundamental basis for its function, and precise determination of its amino acid sequence is critical for advancing our understanding of biological processes. Leveraging high-resolution mass spectrometry and advanced analytical platforms, primary structure sequencing is becoming increasingly rapid, accurate, and comprehensive. MtoZ Biolabs is committed to delivering expert and dependable protein structural analysis services. For protein sequencing, structure verification, or functional research needs, please contact us for customized solutions.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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