Q&A of Protein Sequencing
Q1: Does protein sequencing require the protein to remain active?
A1: No. Protein sequencing focuses on determining the amino acid sequence, not the biological activity of the protein. Whether using Edman degradation or mass spectrometry, the functional state of the protein has no impact on sequencing accuracy. In fact, most sequencing workflows—including reduction, alkylation, digestion, and ionization—disrupt the protein’s native structure and eliminate biological activity. What matters is that the sample is structurally intact, free from significant degradation or contamination.
Q2: How do we choose between Edman degradation and mass spectrometry for protein sequencing?
A2: The choice depends on your sample type and sequencing goals:
Edman degradation is ideal for highly purified proteins (>90%) when the goal is to determine the N-terminal sequence, such as verifying signal peptide cleavage or identifying the starting residue. It reliably reads the first 10–30 amino acids but cannot analyze proteins with blocked or modified N-termini.
Mass spectrometry is better suited for lower-purity samples, mixtures containing multiple proteins, or when the target protein is unknown. It excels at analyzing complex samples, identifying proteins via peptide mass fingerprinting, performing full-sequence analysis, and detecting post-translational modifications. Unlike Edman degradation, mass spectrometry doesn't rely on the N-terminus and offers broader coverage with database-driven, high-throughput capability.
Q3: Can protein bands from SDS-PAGE gels be directly used for mass spectrometry?
A3: Yes, protein bands from SDS-PAGE gels can be used for mass spectrometry, but they must first be excised from the gel, digested with enzymes (e.g., trypsin), and then analyzed. Proper washing and de-staining are necessary to remove contaminants that could interfere with MS analysis.
Q4: What should be done if the target protein is unknown and its sequence is not present in the database?
A4: In this case, de novo sequencing can be used. This method, using mass spectrometry, analyzes peptide fragments and assembles the sequence without relying on a reference database. It is particularly useful for identifying variant proteins or non-natural proteins. Multiple enzyme digestions and high-resolution MS/MS can enhance sequence coverage and accuracy.
Q5: What are the main strategies for protein sequencing, and what research needs are they suited for?
A5: The main strategies for protein sequencing include the following:
1. Bottom-up sequencing
Bottom-up is the most widely used protein sequencing strategy. Proteins are enzymatically digested (e.g., with trypsin) into short peptides, which are then analyzed by LC-MS/MS. Sequence information is reconstructed through database searching or de novo assembly. This method offers high sensitivity and throughput, making it ideal for complex samples, protein mixtures, and large-scale proteomics studies. It supports relative quantification (e.g., via TMT or iTRAQ labeling) and post-translational modification (PTM) analysis. However, because proteins are broken into fragments, it is not suitable for distinguishing isoforms or mapping co-existing PTMs.
2. Top-down sequencing
Top-down sequencing analyzes intact proteins directly using high-resolution mass spectrometry without enzymatic digestion. It preserves the native modification patterns and enables precise mapping of PTMs, isoforms, splice variants, and structural variants. This method is particularly well-suited for studies requiring full structural integrity, such as antibody sequencing, therapeutic protein characterization, or native proteoform profiling. However, it requires highly purified samples, high-performance MS instrumentation, and is typically used for low-complexity samples or single proteins.
3. Middle-down sequencing
Middle-down bridges the gap between top-down and bottom-up. Proteins are digested into medium-sized fragments (typically 3–10 kDa) using non-specific proteolysis. It offers a balance between structural continuity and analytical resolution. It’s advantageous for analyzing antibody heavy chains, repetitive sequences, or heavily modified regions. Middle-down offers better sequence coverage and PTM clarity than bottom-up, while easing some of the technical demands of top-down. It’s suitable when PTM context matters but sample conditions aren’t optimal for full top-down analysis.
Q6: What should be considered during protein sample preparation?
A6: During protein sample preparation, it's crucial to avoid introducing external contaminants, as even trace amounts of foreign proteins can compromise the accuracy of mass spectrometry results. Use clean, contamination-free labware and reagents of MS-grade purity. All solutions should be freshly prepared. Operators must wear gloves and head covers to prevent keratin and other contaminants from affecting the samples.
Q7: Can full-length protein sequencing detect post-translational modifications (PTMs)?
A7: Yes. Full-length protein sequencing using high-resolution mass spectrometry (e.g., Top-down or Middle-down MS) can directly identify and localize PTMs such as phosphorylation, acetylation, and oxidation. Unlike Bottom-up approaches, full-length sequencing preserves structural information surrounding the modification sites, making it especially valuable for studying PTM crosstalk and conformational regulation.
However, PTM detection requires high-resolution instrumentation and careful control of sample complexity and data processing. For low-abundance or multi-site modifications, enrichment strategies are recommended to enhance detection sensitivity and site coverage.
Q8: What Analysis Can Be Performed on Protein Sequencing Results??
A8: Protein sequencing results support a wide range of downstream analyses that help reveal a protein’s structure, function, modifications, and biological roles. Common analyses include:
1. Sequence Alignment: Identify homologous proteins to infer function and evolutionary relationships.
2. Functional Domain & Active Site Prediction: Detect conserved domains and catalytic residues critical for protein activity.
3. 3D Structure Prediction: Model protein structures to understand molecular interactions and conformational dynamics.
4. Protein–Protein Interaction Analysis: Explore interaction partners to map cellular pathways and protein complexes.
5. Post-Translational Modification (PTM) Mapping: Localize modifications such as phosphorylation or ubiquitination and study their functional impact.
6. Expression Profiling: Assess differential protein expression across tissues, time points, or disease states.
7. Pathway Integration: Place proteins into signaling or metabolic pathways to understand their regulatory roles.
8. Evolutionary Analysis: Analyze sequence conservation and divergence to trace functional evolution.
9. Biomarker Discovery: Identify candidate proteins for disease diagnosis, prognosis, or therapeutic targeting.
Q9: How can protein sequencing results be validated?
A9: Common validation methods include:
1. Western blot: Use specific antibodies to confirm the presence of the target protein and compare its molecular weight with sequencing results.
2. Molecular weight comparison: Compare the theoretical molecular weight with SDS-PAGE or MS-measured values.
3. Mutational validation: Introduce specific mutations into expression constructs and verify key amino acid sites via sequencing of the expressed protein.
4. Functional assays: If the target protein has a known function, functional assays (e.g., enzymatic activity or binding) can indirectly support the sequencing results.
Using multiple validation approaches improves the confidence and reliability of the sequencing outcome.
Related Services
How to order?
