Comprehensive Guide to Peptide Sequencing Services: From Principles to Applications
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Accurate identification of low-abundance and multi-modified peptides.
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Enhanced data quality through AI-driven and multi-engine analysis pipelines (MaxQuant, Byonic, Mascot),
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Tailored support from an expert scientific team to address challenges in sequencing complex biological samples.
Peptide sequencing, grounded in high-resolution mass spectrometry (MS) technologies, involves the mass analysis, fragmentation interpretation, and bioinformatics-assisted comparison of peptides derived from proteolytically cleaved proteins. Through this process, the linear arrangement of amino acid residues in proteins or polypeptides is elucidated, thereby determining the primary structure of the target protein. Beyond structural elucidation, peptide sequencing enables precise localization of post-translational modification (PTM) sites, facilitating research into protein function, molecular interactions, and roles within biological systems. This approach underpins critical advances in proteomics, drug discovery, and disease mechanism studies. Therefore, understanding both the theoretical foundations and practical implementations of peptide sequencing is a pivotal step in proteomics research, life science innovation, and biopharmaceutical development.
Core Principles of Peptide Sequencing
1. Proteolytic Digestion
Proteins are enzymatically cleaved into peptides amenable to MS analysis using specific proteases such as trypsin, Lys-C, and Glu-C. The digestion process is precisely controlled in terms of temperature, pH, enzyme-to-substrate ratio, and incubation time to ensure optimal peptide length and sequence coverage.
2. Ionization and Mass Spectrometric Detection
Peptides are ionized via electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI). Subsequently, high-resolution MS platforms (e.g., Orbitrap, FTICR) measure the mass-to-charge ratio (m/z) of peptide molecular ions to acquire accurate mass data for intact peptides.
3. Precursor Ion Selection and Fragmentation (MS/MS)
Selected precursor ions (parent peptides) are subjected to tandem mass spectrometry (MS/MS), where they undergo fragmentation through techniques such as collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), or electron transfer dissociation (ETD). This produces b- and y-ion series, which are critical for reconstructing the peptide sequence.
4. Data Analysis and Sequence Deduction
Fragmentation spectra are interpreted through database searching tools (e.g., Mascot, MaxQuant) or de novo sequencing algorithms to infer the peptide’s amino acid sequence. Simultaneously, PTM sites can be identified with high confidence.
Complete Experimental Workflow of Peptide Sequencing
The experimental pipeline for peptide sequencing comprises the following eight steps:
1. Sample Preparation and Protein Extraction
(1) Proteins are extracted using lysis buffers containing buffering salts, detergents, and reducing agents, in combination with physical disruption methods such as ultrasonication and mechanical homogenization.
(2) Disulfide bonds are disrupted via reduction with DTT and subsequent alkylation using IAA to ensure full denaturation of proteins.
(3) Protein concentration is accurately quantified using methods such as BCA, Bradford, or UV absorbance assays.
2. Proteolytic Digestion
(1) Either single-protease (e.g., trypsin) or multi-enzyme (e.g., trypsin + Lys-C, Glu-C) strategies are employed.
(2) Standard digestion conditions include 37°C, pH 7.8–8.0, for 12–16 hours.
3. Peptide Purification and Desalting
(1) Solid-phase extraction (SPE) with C18 cartridges is used to remove salts and contaminants.
(2) For complex samples, further purification may involve gel excision or SDS-PAGE.
4. Liquid Chromatography (LC) Separation
(1) Peptides are separated based on hydrophobicity using nano-flow LC coupled with C18 reversed-phase columns.
(2) Optimized gradients (water-acetonitrile mixtures with acid additives) enhance resolution and sensitivity.
5. Ionization and Mass Spectrometry Detection
(1) LC-ESI-MS/MS is conducted in an online configuration to enable high-throughput analysis.
(2) High-resolution instruments such as Orbitrap or FTICR deliver mass accuracy at sub-ppm levels, enhancing data reliability.
6. Precursor Ion Selection and Fragment Generation
(1) Techniques such as dynamic exclusion and automatic gain control (AGC) are utilized to improve detection of low-abundance peptides by minimizing redundancy from dominant ions.
(2) Fragmentation modes are selected based on analysis objectives: CID for general use, HCD for richer fragmentation patterns, and ETD for preserving labile PTMs.
7. Data Acquisition and Sequence Interpretation
(1) Database search engines match experimental spectra to theoretical fragments with tight mass tolerances (MS1: ±10 ppm, MS2: ±0.02 Da).
(2) De novo sequencing is applied to deduce novel peptide sequences directly from MS/MS data.
(3) Multiple software tools (e.g., Mascot, MaxQuant, Byonic) and AI-assisted algorithms (e.g., Prosit) are integrated to enhance sequence identification and confidence.
8. PTM Localization and Multi-Dimensional Validation
PTM sites such as phosphorylation and glycosylation are accurately localized using information from retention time, fragmentation patterns, and isotope distribution profiles.
Application Domains and Emerging Trends in Peptide Sequencing
1. Application Domains
(1) Proteomics Research
Systematic characterization of protein profiles in samples such as cells, tissues, and plasma.
(2) Post-Translational Modification Analysis
Precise mapping of critical PTMs, including phosphorylation, acetylation, glycosylation, and ubiquitination, to elucidate regulatory mechanisms.
(3) Neoantigen Discovery
Identification of novel antigenic peptides for use in cancer immunotherapy and vaccine design.
(4) Biomarker Development
Screening of disease-associated peptides for early detection, diagnostics, and therapeutic monitoring.
(5) Biopharmaceutical Quality Control
Verification of protein drug purity and batch-to-batch consistency.
2. Emerging Trends
(1) Single-Cell and Spatial Peptide Sequencing
Enabling proteomic analysis at the level of individual cells and spatial tissue architecture.
(2) Multi-Modal Technology Integration
Combining tandem MS with mass spectrometry imaging (MSI) and in situ approaches (e.g., DESI-MS) to achieve comprehensive protein mapping.
(3) AI and Deep Learning Integration
Enhancing the precision and speed of peptide identification and PTM localization in complex samples through advanced computational tools.
As a leading provider in the field of proteomics and peptide sequencing, MtoZ Biolabs leverages state-of-the-art high-resolution mass spectrometry platforms (e.g., Orbitrap Fusion Lumos, Q-Exactive HF-X, MALDI-TOF/TOF), along with proprietary workflows for sample preparation and data analysis. The company delivers end-to-end peptide sequencing solutions to global academic and industrial clients, including:
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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