The Byos™ disulfide bond (S-S) workflow provides a robust platform for the identification and quantification of disulfide-bonded, trisulfide-bonded, and cross-linked (Xlink) peptides. Designed to reduce manual effort and increase confidence in results, the workflow integrates automated validation, streamlined data review, and customizable reporting.
Key capabilities include:
- Comprehensive workflow configuration for both S-S and Xlink analyses
- Automated Disulfide Validator to ensure accurate assignment and reduce false positives
- Advanced data visualization with Xlink labeling for efficient inspection of complex results
- Customizable reporting tools for consistent, reproducible documentation of findings
Workflow set-up
Byos software is vendor-independent and supports all raw file types, which can be loaded directly into the Samples tab
Protein sequences or chains can be loaded directly into the Sequences tab.
Defining Expected S-S Linkages:
- By default, the S-S workflow is configured for IgG molecule analysis. To analyze non-IgG proteins, simply deselect the IgG option.
- When working with IgG molecules, specify the heavy chains and light chains using the dropdown menu.
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The software will automatically populate the Disulfide links (expected) table with the corresponding disulfide bond combinations. This table can be edited to enter user defined expected S-S linkages.
Note: The software will automatically search for any combination of S-S linkage. Peptides matching the listed S-S bond patterns are categorized as Expected, whereas any other combinations are classified as Shuffled.
Workflow set-up
Defining Xlink search type:
The S-S and Xlink settings are located in the Processing nodes tab as part of the Byonic (MS2) search parameters.
To configure this section:
- Select either Disulfide, Trisulfide or Crosslink options.
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By default, the workflow is set to search for cross-links using DSS. To use other chemical cross-linkers (e.g., DSSO, DSBU), configure them under Enable Crosslink Custom.
For instructions on parameter settings for cross-linked peptides, including support for cross-linking between multiple amino acid residues, see this article.
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The values in the For FASTA Proteins option indicates the protein or chain position number in the FASTA file. For example...
“1,2” searches for both inter- and inter-chain S-S bonds between protein 1 and 2.“1,2;3”, searches for S-S bonds between proteins 1 and 2. For protein 3, search for inter- and intra-peptide S-S bonds of protein 3, but not search for between proteins1,2 and 3.
Byonic parameters for S-S and Xlink searches
Support for Project Review and Validation
Once the project has been created the user will have access to the inspection view and a report. The inspection view is used to validate S-S and Xlinked peptides before they are passed to the report.
The peptides table contains various filtering options that allow for more streamlined data review and validation.
Using a combination of the Search filter and Column filters can narrow down peptides for review.
- The search filter allows for the selection of S-S and Xlinked peptide pairs
- By filtering on the Xlink Class column, Expected disulfides can be filtered out to focus review time on “Shuffled” disulfides.
- In addition to manual validation, Byos includes a Disulfide Validator* for automated peptide validation. Peptides that meet the pre-defined criteria are labeled as clear/no flags in the comment column, while peptides that do not meet the criteria are flagged as either critical or warning, helping to prioritize review and ensure data quality
Filtering options in the peptides table
*The disulfide validator requires an advanced command to be entered into the workflow at the project creation stage. This advanced command can be copied from below and pasted into the Advanced Configuration section of the workflow, under the Byologic node. Full details can be found in the Peptide Analysis Manual (located in user guides):
[Byologic]
DisulfideValEnabled=true;
Disulfide validator options
Shortcut keys allow peptides to be quickly labeled as true positive (t), false positive (f), or other categories. Once validation is complete, false positives can be easily removed using the search filter.
Assigning and filtering peptides based on validation type
Automated Reporting for S-S Peptides
Once validation is complete, the results can be viewed in the Byos S-S report, which contains a series of tables and charts. The S-S report template is customizable and contains:
Filters including Xlink-type can group and display disulfides, trisulfides, and free thiols.
- Bar charts comparing XIC areas for total expected vs. shuffled S-S bonds (a) and a bar chart of total disulfides vs. trisulfides (b).
- A table grouping peptides, disulfide bonds, trisulfide bonds, and others based on cysteine residues.
- A table showing the relative abundance of all S-S bonded peptides.
Byos delivers an end-to-end workflow for S-S and Xlink analysis, enabling scientists to accelerate analysis while making decisions with confidence.
For more information contact us at support@proteinmetrics.com