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:
target_protein=example_protein
where example_protein can be any substring that matches the *.fasta header 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.
Specifying how homologous peptides are annotated in the protein coverage table
The user can determine whether a homologous peptide will be included in the coverage map of either the same protein or any additional lower scoring proteins in the search results.
To do this, the following advanced command should be entered at project creation to the Quant (Byologic) node of the workflow:
[ProteinCoverage]
AddDuplicates = 2The number used will determine how duplicates are treated:
0 - no duplication
1 - duplication within the same protein
2 - duplication across all proteins
3 - duplication within the same protein applying protease specificity
4 - duplication across all proteins applying protease specificity
If we consider a protein chain that contains multiple occurrences of the sequence LTISK. When AddDuplicates is set to 0, this results in only the first occurrence of the sequence to be included in the coverage map.
When AddDuplicates is changed to 1 this results in all occurrences of the sequence being included in the coverage map.
If the AddDuplicates value is changed to 3 then the duplicate peptide will only be included in the coverage map if the flanking N and C-terminal residues satisfy the specified protease in the samples table.
The protease used should be specified in the samples table at project creation. Byos supports simultaneous proteases that can either be selected from the options in the drop down menu, or by creating a custom protease or a combination.
If we now consider a bispecific antibody, in which both heavy chain 1 (HC1) and heavy chain 2 (HC2) contain the sequence LTISK, when AddDuplicates is set to 0 the peptide only appears in the coverage map for the highest scoring chain (HC 1). When the value is set to 2 the peptide appears in the coverage map for both chains.
If AddDuplicates is set to 4 the protease specificity (specified in the samples table) will be used to ensure that each assignment satisfies the N and C-terminal protease cleavage sites.
Utilizing this system empowers users to control how repetitive sequences are annotated and reported in their peptide mapping experiments.
Reporting options
It's possible to report where a duplicated peptide sequence occurs, including the names and locations of the associated protein(s). To do this, go to the pivot table tab in the report, open the File menu, and choose Presets → Pre Pivot Columns → TiskRowClone.prepvt. Then name the new column something like "Duplicates."
Once this process has been performed the table will show both protein names along with the position of the peptide and any modifications.
Reassigning homologous peptides
Lastly, to ensure that a peptide is assigned to the same protein sequence across all samples, the user can utilize the AdjustPrimaryProtein advanced command:
[Byologic]
AdjustPrimaryProtein = true;When enabled, any inconsistent protein assignments would be corrected by assigning the peptide in all samples to the highest-ranking protein. If HCP searches against multiple samples yield different primary proteins for a given sequence, this option can be used to synchronize the samples.