Database Assisted Shotgun Sequencing (DASS) Technology

Creative Biolabs is gladly to show a series of unrivalled de novo antibody

sequencing services for research, diagnostic and therapeutic industries. To
overcome the current drawback of sequencing based on traditional methods,
Creative Biolabs has developed the proprietary Database Assisted Shotgun
Sequencing (DASS) technology, which based on our next generation antibody
sequencing platform. Our services consist of variable region, variable plus
leader region and 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
QC analysis
IND/NDA application
Service Specifications
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. If we do not reach 100 % coverage, you will not be
charged.
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
remain 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 homologue in the
database.
Isobaric amino acids
In contrast to other MS based methods we can discriminate most isobaric

amino acid combinations. E.g.:

W can be distinguished from GE, AD and SV (by mass difference) .
R can be distinguished from GV (by mass difference) .
Q can be distinguished from GA and K (by fragment spectra and mass
difference) .
N can be distinguished from GG (by derivatization and fragment spectra) .
Leucine and isoleucine cannot be distinguished. However, most of these
positions can be determined using the corresponding germline sequence (see
Fig. 1). As antigen binding is mostly mediated by salt bridges and hydrogen
bonds the impact of Leu/Ile is usually negligible.
More details about de novo sequence.