Peptide mapping of biotherapeutics such as bispecific antibodies and AAV proteins can be challenging due to the high level of homology found between different chains (in the case of antibodies) or viral proteins (as with AAVs). This results in duplicate peptides that make it difficult to correctly represent protein coverage.
In cases where multiple proteins within a database share regions of sequence homology, the Byonic™ MS/MS search engine will generate a peptide spectrum match (PSM) for every protein in which a given peptide is identified. Once the protein ranking is completed, each peptide will be attributed to the protein with the highest score. There are now two ways to change this behaviour in Byonic:
First, the user can tell Byonic to exclude homologous peptides when calculating the protein score, meaning that only unique peptides will contribute to the protein score. This provides an unbiased mechanism for assigning homologs to the appropriate protein. To turn the feature on, add the following line to the modifications edit box:
dont_score_homologous_peptides = 1
Second, it’s now possible to prioritize the protein assignment of a homologous peptide to a specific protein. The prioritized targets are defined in the Byonic modifications window. To turn the feature on, add the following line to the modifications edit box:
where example_protein can be any substring that matches the*.fastaheader of the target protein(s).
Reporting protein occurrence
Peptide analysis in Byos® (legacy Byologic) uses smart features that give the user control over how homologous peptide sequences are used to represent protein coverage.
A "protein occurrence" column can be added to the peptides table to indicate if the peptide is present in one or more proteins. Following on from this, a protein summary table can be opened from the peptides table allowing the user to see details of all possible matches.