Antibody Sequencing Services

Antibodies possess well-defined and highly specific targeting and binding capabilities, playing an increasingly important role in basic research, disease diagnosis, and clinical therapy. Currently, antibody sequencing services at the amino acid level primarily rely on mass spectrometry-based de novo sequencing technology. De novo protein sequencing is an innovative technique that does not depend on any known sequences or protein database information, directly determining the amino acid sequence of antibodies or proteins. This technology is particularly effective for identifying novel or previously unknown antibody sequences. Antibody sequence information is crucial for understanding the structural basis of antigen binding, how somatic hypermutation controls affinity maturation, and constructing antibody libraries to comprehensively study adaptive immune responses in health and disease. Furthermore, it holds significant importance in the production and quality control of antibody-based therapeutics and diagnostic reagents.

 



Peng, W W. et al. Journal of Proteome Research,2021.

Figure 1. One of The Applications of Antibody Sequencing: Quality Control of Antibody Reagents.

 

Services at MtoZ Biolabs

The mass spectrometry (MS)-based antibody sequencing services leverage mass spectrometers to acquire peptide amino acid sequence mass information via tandem mass spectrometry (MS/MS) spectra. Through de novo sequencing algorithms, peptide sequences are directly deciphered from MS/MS spectra and assembled to obtain the complete amino acid sequence of the antibody. The technical workflow of MS-based antibody sequencing services include sample preparation → protein digestion → peptide separation → mass spectrometry detection → data analysis.

 

MtoZ Biolabs has developed proprietary software for mass spectrometry antibody sequence analysis, equipped with self-developed raw data computation methods. By employing a broad-to-narrow approach, we ensure that no valuable data is overlooked while achieving precise antibody sequence identification. The antibody sequencing services provided by MtoZ Biolabs encompass two key directions: MS-based de novo sequencing and graph theory-based sequence assembly.

 

1. MS-Based De Novo Sequencing Service

MS-based antibody sequencing services primarily rely on de novo sequencing technology. Using algorithms based on deep learning sequence models, the amino acid composition and arrangement of peptides are directly deciphered from MS/MS spectra. De novo sequencing enables precise differentiation of amino acids within peptides and quantitative analysis of their abundance levels within protein samples.

 

MtoZ Biolabs employs Thermo Fisher's Orbitrap series mass spectrometers in combination with Nano-LC systems to ensure high-precision and high-sensitivity data acquisition. This combination of technologies allows for the accurate determination of amino acid composition, sequence arrangement, and abundance levels of peptides, delivering reliable results for full-length antibody sequencing.

 

2. Graph Theory-Based Sequence Assembly Service

In addition to MS-based de novo sequencing, MtoZ Biolabs utilizes graph theory-based sequence assembly algorithms. By applying deeply covered assembly rules, we ensure the accurate reconstruction of full-length antibody sequences.

 



Graaf, S C D. et al. mAbs,2025.

Figure 2. Overview of Mass Spectrometry-based Antibody Sequencing Methods.

 

Service Advantages

1. Advanced Analytical Platform

MtoZ Biolabs has established advanced antibody sequencing services platform, ensuring reliable, rapid, and high-precision analysis services.

 

2. Transparent Pricing

Our pricing structure is transparent, with no hidden fees or additional fees.

 

3. High Data Quality

Deep data coverage and strict data quality control. The AI-driven sequencing platform ensures full-length sequencing coverage and accuracy, and provides customers with comprehensive data reports.

 

Sample Submission Suggestions

1. Sample Types

Antibody purity >90%, with a total amount >100 µg.

 

2. Sample Amount

For antibody solutions, buffer details should be noted. For gel samples, each lane must contain at least 10 µg, with a minimum of 6 lanes submitted.

 

3. BSA

Excess BSA in samples should be avoided. If present, the concentration must be noted.

Note: If your sample has specific requirements or does not meet the above conditions, please contact us for customized support.

 

Applications

1. Antibody Discovery

Antibody sequencing services detect individual-specific antibody proteins that are overexpressed in some immune-related diseases and can be used as disease markers.

 

2. Antibody Drug Quality Control

Antibody sequencing technology can identify mutated amino acids and degradation sites in antibody drugs.

 

3. Antibody Production Monitoring

Antibody sequencing analysis can determine whether the antibody product is expressed according to the expected sequence.

 

4. Antibody Reverse Engineering

Due to the opaque information of antibody product suppliers, recombinant antibodies can be used to maintain antibody stability and reduce costs by sequencing antibody proteins.

 

5. Antibody Engineering

Antibody sequencing services support the identification of key amino acids that influence stability, affinity, and activity, aiding in antibody optimization.

 

Case Study

1. Using Antibody Sequencing Technology to Determine Antibody Sequence and Reconstruct the Crystal Structure

Anti-FLAG-M2 antibodies are widely used to detect and purify FLAG-tagged proteins. The crystal structure and sequence information of anti-FLAG-M2 antibodies can be queried from public databases, but the key amino acid information of the antibody heavy chain is erased—use 'A' Replace the original amino acid information. Researchers employed multiple enzymatic digestion methods to obtain peptide fragments of the antibody and used HCD and ETHCD fragmentation modes to collect MS data of the peptides. This approach achieved full-sequence coverage of the monoclonal antibody trastuzumab. By performing de novo sequencing and sequence assembly on the widely used anti-FLAG M2 mouse monoclonal antibody, the complete anti-FLAG M2 antibody sequence was determined. Furthermore, the antibody's 3D crystal structure was reconstructed using the experimentally determined sequence.

 



Peng, W W. et al. Journal of Proteome Research,2021.

Figure 3. Mass Spectrometry-based De Novo Sequence of Mouse Monoclonal Anti-FLAG-M2 Antibody.

 



Peng, W W. et al. Journal of Proteome Research,2021.

Figure 4. Validation of Anti-FLAG-M2 Sequence Based on Mass Spectrometry.

 

Deliverables

1. Comprehensive Experimental Details

 

2. Materials, Instruments, and Methods

 

3. Quality Control Assessment

 

4. Antibody Sequence

 

5. MS/MS supporting evidence of antibody sequence

 

6. Raw Data Files

 

MtoZ Biolabs, an integrated Chromatography and Mass Spectrometry (MS) Services Provider, provides advanced proteomics, metabolomics, and biopharmaceutical analysis services to researchers in biochemistry, biotechnology, and biopharmaceutical fields. Our ultimate aim is to provide more rapid, high-throughput, and cost-effective analysis, with exceptional data quality and minimal sample consumption. Free project evaluation, welcome to learn more details!