Background

Creative Biolabs recently has unprecedentedly achieved the precise identification of leucine and isoleucine by mass spectrometry, advancing breakthrough science on protein/antibody sequencing in research, diagnostic, and therapeutic industries. The discrimination of leucine and isoleucine has long been the key technical difficulties of de novo antibody sequencing. However, Creative Biolabs just took a massive leap forward into the new era of this field, with unchallenged ability to achieve 100% accuracy in antibody sequencing by precisely distinguishing between leucine and isoleucine, based on the proprietary Database Assisted Shotgun Sequencing (DASS) technology.

Our services consist of variable region, variable plus leader region, as well as full-length heavy- and light-chain antibody sequencing for all species, isotypes, and allotypes. Purified monoclonal antibodies in multivalent forms can be sequenced with 100% coverage of the desired regions as well as excellent accuracy. Numerous successful cases from Creative Biolabs have confirmed our qualification to provide antibody sequencing with 100% accuracy and satisfaction guarantee to meet our customers' needs.

Our world-leading DASS system provides reliable supporting proof for:

• Confirmation & validation of produced mAb, protein and enzyme
• QC analysis
• IND/NDA application

General Workflow for Antibody Sequencing

Based on our proprietary technology for de novo sequencing of antibodies and our expertise in protein mass spectrometry and informatics, our standard procedures for service include the following steps:

Fig. 1 De Novo Antibody Sequencing Workflow.

• Antibody protein is initially digested into overlapping peptides using various trypsin
• Digestion with LC-MS/MS, then fragmentation optimization
• Overlapping peptides are characterized by de novo peptide sequencing
• Automated antibody sequence assembly using characterized peptides
• Data analysis is performed using state-of-the-art computational algorithms that efficiently derive the amino acid sequence of the digested peptide

One-Stop De Novo Antibody Sequencing Services

Sequencing of the V and J and C Segments by DASS

The V and J and C gene segments of antibodies are available in public databases. However, during the maturation of an antibody, the B cell introduces hypermutations into the sequence to optimize the affinity. Our mapping algorithm is error tolerant and can match the "mutated" peptides to the corresponding germline, reliably.

Due to the high number of peptides, we get sequence information for EVERY peptide bond in the antibody. Typically, 20-70 different MS/MS spectra are generated for each amino acid (AA) position. Hence, even the hardest sequences of proline and arginine-rich peptides can be resolved. As the order of all amino acids is clear, there is no need for time-consuming techniques like Edman sequencing.

De Novo Sequencing of the CDR3 Region

While the CDR3 of the light chain is mostly encoded by the germline sequences, the CDR3 of the heavy chain is usually not available in databases. It is encoded by the so-called D-segments, but these are modified by nucleases and terminal transferases. Typically, only 1-4 AA of a D-segment remains in the matured antibody. The rest of the D-segment is “artificial” and has to be sequenced de novo.

Our method generates many overlapping peptides during the fragmentation process, enabling us to sequence very long stretches of unknown amino acids. The high quality of MS/MS spectra in combination with intelligent data mining, allows us to read the CDR3 like a book. The technique is so powerful that we were able to sequence a 20 kDa protein, which had no homolog in the database.

Sequencing of Fluorochrome mAbs, IgMs, and other Non-Standard Antibodies

• We have over 90% chance to get the sequence from tiny amount of your precious sample (e.g. 20 µg).
• We can enrich even very dilute sample (e.g. western blot antibody) for sequencing.
• Our method is usually not affected by ligands (FITC, Biotin, Alexa) coupled to antibodies. Larger protein ligands (PE, APC) make sequencing slightly more challenging. However, this can be compensated by using a higher protein concentration, because sequencing quality is dependent on sample amount.
• IgMs can usually be sequenced like normal IgGs. Slightly more sample may be required. As the constant region is modified by several glycans, which cannot be cleaved by PNGase F, we can only guarantee complete coverage for the variable part of the antibody.
• Our method works with all antibody formats from all species (mouse, rat, hamster, llama, rabbit, etc.; Fab, scFv, etc.).
• Some mAbs are coupled to matrices. This is compatible with our sequencing method, but more samples may be required.
• Many older hybridomas may produce 2 light chains (one is the kappa-chain of myeloma MOPC 21). These mixtures can usually be sequenced, even when the MOPC 21 kappa chain is in two-fold molar excess.
• We also provide DASS-Based Protein and Enzyme Sequencing service including full end to end sequencing, determination of post-translational modifications, intact mass, N-glycosylation sites, and artificial tags, etc.

Isobaric Amino Acids

In contrast to other MS-based methods, we can discriminate most isobaric amino acid combinations, which including but not limited to:

Fig. 2 Sample Sequencing Results.

• Leucine can be distinguished from Isoleucine.
• W can be distinguished from GE, AD and SV (by mass accuracy).
• R can be distinguished from GV (by mass accuracy).
• Q can be distinguished from K (by mass accuracy).
• N can be distinguished from GG. Our DASS technology ensures bond breakage between GG. In addition, we use our proprietary derivatization technology for asparagine to ensure that it is not GG.
• Q can be distinguished from GA. Our DASS technology ensures bond breakage between GA. In addition, we use our proprietary derivatization technology for glutamine to ensure that is not GG.

Sequence Validation Guarantee

As we are aware that your projects have tight timelines, we validate the sequences to ensure that your antibody will be functional with the first attempt.

(1) The masses of the reduced light- and heavy-chain will be determined to validate the determined sequences. The light chain sequence must match within a +/- 1.2 Da range. For the heavy chain, +/- 1.8 Da is allowed. If the determined sequences do not match with the protein mass, you will not be charged. (Please note that this deviation is less than the weight of two protons).

(2) Each mutation in the antibody will be validated by at least two meaningful spectra. This ensures that your sequence does not contain isobaric exchanges. Do not rely on peptide maps alone. [Isobaric exchanges are exchanges of identical mass like GG=N, YM=FM (Ox). The peptide maps used by some competitors are often not sensitive to this kind of error].

Coverage Guarantee

If we do not reach at least 5× coverage for the complete V(D)J-region, you will not be charged. You can also book our 10× service. We will not stop to sequence unless we have 10× coverage for V(D)J.

Highlights from Creative Biolabs

The one and only: Discrimination of leucine & isoleucine.

Fast: Sequencing can be accomplished within 10 working days. Additional 2~3 weeks would be needed if the discrimination of leucine & isoleucine is required.

Sensitive: Full coverage can be obtained from 50-100 μg of protein.

Robust: We guarantee 100% coverage for the VJ and VDJ region.

High-throughput: Our DASS method is high-throughput compatible. We would be pleased to plan your project with 100, 200, 500 or more antibodies.

Professional: Highly experienced Ph.D. scientists in charge of every project with a specialized team for quality control in every step of the process; expert R&D team dedicated to the optimization of protocols to deliver data with enhanced quality.

Integrity: Custom services including but not limited to antibody designing, cloning, de novo sequencing and analyzing, as well as in vitro optimization to fulfill your needs.

If you are interested in our DASS technology, please feel free to inquire us for more details.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.