Homology bias and IgBLAST
Because IgBlast relies on homology-based searches, sequences of novel antibodies or antibodies from not routinely researched species, or species with poor annotated genomes, may be difficult to fully identify, or worse may be impacted by bias. This is why de novo protein sequencing is important as it allows to sequence an antibody that is novel or from an uncommon species
Distinguishing between isoleucine and leucine using IgBLAST
Furthermore, with de novo sequencing, users can distinguish between same-mass residues such as Isoleucine (Ile, I) and leucine (Leu, L) that may be confounded in database searching in proteomics databases. An I/L identity in a reference sequenced provided by IgBLAST is based on the germline gene sequence, which may be reliable if the sequence has been verified consistently in a statistically significant way (e.g., reference sequences in SWISS-Prot).
However, if an I vs L decision must be made through de novo protein sequencing of a novel or engineered sequence or from a sequence of a not-well annotated genome, scientists may not always be able to rely on a reference sequence from a database. Thus to confirm the identity of isobaric residues I and L, de novo sequencing is critical. You can read more about how I/L elucidation is effected here.
I and L determination is especially important for antibodies since they often present in the complementarity-determining region (CDR) of antibodies and are important to elucidate for binding. Databases like IgBLAST may also not be able to give a reference sequence of CDR-H3, which is the most unique portion of an antibody sequence, and thus highly heterogeneous even within the same species and animal.
CDR diversity can only really be deconvoluted via de novo sequencing
Though CDR1 and CDR2 are encoded in the V segment, CDR3 is somatically generated via recombination of the V and J segments (light chain CDR-L3) or recombination of V, D, and J segments (heavy chain CDR-H3). IgBLAST stores reference germline genes which do not take into account insertions, deletions and/or rearrangements from V(D)J recombination which are vital to antibody sequence diversity.
If you think you would benefit from the de novo capabilities of next generation protein sequencing, reach out to discuss more with our scientists.