Protein Full-Length Sequencing Workflow: From Sample Preparation to Data Interpretation
Protein full-length sequencing, including top-down and de novo protein sequencing approaches, has emerged as a precise and comprehensive strategy for acquiring complete protein sequence information. As proteins are central executors of biological function, their amino acid sequences are critical for elucidating structural features, functional roles, and post-translational modifications (PTMs). Traditional protein identification methods often rely on database matching, which limits their utility in characterizing unknown proteins or novel isoforms.
This article presents a systematic overview of the protein full-length sequencing workflow—from sample preparation to data interpretation—highlighting key steps and optimization strategies to aid researchers in applying this advanced technique effectively.
Sample Preparation: The First Determinant of Sequencing Quality
Protein full-length sequencing demands exceptionally high sample quality. Key preparatory steps include:
1. Protein Extraction and Purification
(1) Source diversity: Target proteins can be extracted from cells, tissues, or recombinant expression systems.
(2) Purity requirement: A purity of >90% is recommended to minimize interference from contaminating proteins.
(3) Common methods: SDS-PAGE gel excision and affinity purification (e.g., His-tag, FLAG-tag) are widely employed.
2. Desalting and Buffer Optimization
(1) High salt concentrations and buffering agents like Tris can suppress ionization efficiency in mass spectrometry. These components should be removed using dialysis, gel filtration, or C18 solid-phase extraction.
(2) Final reconstitution should be performed in MS-compatible buffers, such as 0.1% formic acid aqueous solution.
3. Concentration and Loading Control
Protein concentration should be maintained between 0.5–2 μg/μL to ensure consistent injection. Typically, 5–20 μg of protein is required per sequencing run.
Enzymatic Digestion Strategy: Designing for Complete Sequence Coverage
To enhance coverage and sequencing accuracy, protein full-length sequencing often integrates multiple enzymatic digestion strategies:
1. Single-Enzyme Digestion (e.g., Trypsin)
Produces predictable peptide fragments, suitable for database-based identification.
2. Non-Specific Digestion (e.g., Proteinase K)
Disrupts cleavage-site bias, enabling effective de novo sequencing.
3. Multi-Enzyme Digestion
Parallel digestion with different proteases generates complementary datasets.
MtoZ Biolabs has developed an optimized multi-enzyme fusion protocol that significantly improves sequence coverage and variant detection, making it highly effective for the complete characterization of complex proteins.
Mass Spectrometry Analysis: The Core Platform for High-Resolution and High-Sensitivity Detection
Protein full-length sequencing relies on cutting-edge mass spectrometry instrumentation:
1. Common Analytical Platforms
(1) Orbitrap Fusion Lumos: Offers high resolution and mass accuracy, ideal for de novo sequencing.
(2) timsTOF Pro 2: Incorporates ion mobility for deeper peptide coverage.
(3) EvoSep One coupled with nano-LC: Enables high-throughput analysis with minimal sample consumption.
2. Fragmentation and Analysis Modes
(1) HCD/CID/ETD tandem fragmentation: Ensures broad coverage of both hydrophilic and hydrophobic peptides, as well as PTMs.
(2) MSⁿ mode: Enhances precision in resolving isomeric sequences.
At MtoZ Biolabs, through precise optimization of MS parameters, we consistently achieve >90% average sequence coverage and >95% accuracy at N- and C-terminal regions.
Data Interpretation and Validation: From Raw Spectra to Amino Acid Sequences
1. De Novo Sequence Interpretation
Dedicated software such as PEAKS Studio, pNovo, and Novor is employed for de novo peptide assembly. Cross-platform comparisons and manual validation are performed to ensure accuracy, particularly in key regions.
2. Sequence Assembly and PTM Identification
Assembled peptide sequences are used to reconstruct the full-length protein, while also identifying PTMs such as phosphorylation, acetylation, and methylation.
3. Database Matching and Application
Reconstructed sequences are matched against UniProt or custom databases to confirm mutations and novel isoforms. Applications include antibody sequence verification, vaccine epitope discovery, and quality control of recombinant proteins.
Application Value and Advantages of MtoZ Biolabs
Protein full-length sequencing has demonstrated broad applicability in areas such as:
1. Antibody sequence confirmation and optimization.
2. Quality control of recombinant proteins.
3. Characterization of proteins in non-model organisms.
4. Analysis of protein variants and splice isoforms.
At MtoZ Biolabs, we integrate multi-platform MS technologies with advanced multi-enzyme digestion workflows to provide high-coverage, high-accuracy, and rapid-turnaround protein full-length sequencing services. Our platform has successfully supported dozens of high-impact projects in drug development, immunotherapy, and synthetic biology.
Protein full-length sequencing is a powerful tool for decoding structural and functional diversity in proteins. While technically demanding, with careful optimization of each workflow stage—from sample preparation to data interpretation—researchers can obtain highly accurate and reliable sequence information. If you are seeking a trusted partner for advanced protein sequencing, we welcome you to collaborate with MtoZ Biolabs. We are committed to empowering your scientific discoveries.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
Related Services
How to order?
