Ten Q&As on De Novo Protein Sequencing

Protein de novo sequencing is a groundbreaking technology that directly determines amino acid sequences without relying on any known sequences or protein databases. It is crucial in medical diagnosis and treatment. To address common questions and concerns regarding sample requirements and details for de novo protein sequencing, MtoZ Biolabs has compiled the following frequently asked questions for your reference.

 

Q1: What Distinguishes Protein De Novo Sequencing from Edman Degradation Sequencing?

A: Protein de novo sequencing uses algorithms to directly deduce peptide sequences from ion information in mass spectrometry spectra. Edman degradation is generally suitable for peptides with 15-20 amino acids and requires high sample purity (at least 97%). It also involves proteolysis, peptide separation, purification, and step-by-step peptide testing. Additionally, the time and cost associated with Edman degradation sequencing for a monoclonal antibody are relatively high.

 

Compared to traditional Edman degradation, mass spectrometry-based de novo sequencing is more efficient, high-throughput, and cost-effective. Even with known protein sequences, de novo sequencing can identify new protein variants, including those resulting from unknown mutations, splicing, and various post-translational modifications, providing comprehensive information for monoclonal antibody sequences.

 

Q2: What Sample Types Are Suitable for Protein De Novo Sequencing?

A: Protein de novo sequencing is independent of protein size and length. Monoclonal antibodies, Fab/Fc fragments, bispecific antibodies, polyclonal antibodies, recombinant proteins, peptides, fluorescently labeled antibodies, and cross-linked antibodies can all be sequenced de novo.

 

Q3: Can Fluorescently Labeled or Cross-Linked Antibodies Be Sequenced De Novo?

A: Proteins cross-linked on beads or fluorescently labeled proteins, such as flow cytometry antibodies labeled with FITC, Cyc5, PE, etc., can also be sequenced de novo.

 

Q4: What Are the Sample Requirements for Protein De Novo Sequencing?

A: The standard sample amount for protein de novo sequencing is 100 µg, with a purity of over 90%. Protein purity significantly impacts sequencing results; impure antibody proteins, homologous proteins, and BSA can interfere with sequencing and affect experimental outcomes.

 

Q5: Do Buffer Components in the Sample Affect Sequencing Results?

A: The recommended sample buffer for protein de novo sequencing is PBS or Tris buffer. If the sample contains glycerol, BSA, detergents, salts, etc., MtoZ Biolabs has methods for purification, ensuring these components do not affect sequencing results.

 

Q6: Does Glycosylation and Post-Translational Modification of Samples Affect Sequencing? 

A: Potential post-translational modifications and glycosylation are considered during data analysis and do not affect the sequencing results.

 

Q7: What Instruments Are Used for Protein De Novo Sequencing?

A: Based on the world's most advanced mass spectrometry instrument, Obitrap Fusion Lumos, and combined with rich experience in bioinformatics analysis, MtoZ Biolabs has established a whole new generation of de novo sequencing platform that can achieve accurate sequence analysis of monoclonal antibodies and proteins, independent of any database searching. Besides, MtoZ Biolabs will continue to introduce more cutting-edge instruments to provide superior services.

 

Q8: What Does the Protein De Novo Sequencing Report Include?

A:

1. Complete amino acid sequence, including heavy and light chains, constant and variable regions for antibodies.

2. Molecular weight verification, comparing the measured molecular weight with the theoretical sequence to verify accuracy.

3. Peptide coverage map of the complete sequence, with each site supported by over ten different peptides.

4. Secondary mass spectra supporting the antibody's variable region peptides.

5. Confidence analysis of I/L identification.

 

Q9: How Accurate Are the Results of Protein De Novo Sequencing?

 A: The results ensure full sequence coverage and 100% accuracy, with each amino acid site supported by over ten different peptides and strong fragment ion peak evidence. The accuracy is verified through software algorithms and manual inspection.

 

Q10: What Is the Turnaround Time for Protein De Novo Sequencing?

A: Conform to the requirements, the standard service time from initial testing to report delivery is one week.