Byos Digested Oligonucleotide provides a vendor neutral solution for the analysis of LC-MS/MS digested oligonucleotide data including:
- Sequence Database Oligo Spectrum Matching (Byonic-Oligo)
- Sequence coverage mapping
- Confident isomer assignment
- Detection and localization of modifications
- Wildcard searching for modification discovery
- XIC Plotting and MS1 Quantitation
- Interactive data review tools and visualizations
- Automated and customizable reporting
This article provides workflow setup instructions. Check out the Byos Digested Oligonucleotide Tutorial Video series for more information. You can always reach out to support@proteinmetrics.com with questions and for more information.
NOTE:
- This Quick Start Guide was written for Byos version 5.6. Most of the concepts will apply to other Byos versions, but there may be differences as we are actively improving this workflow.
1. Open the Byos Digested Oligonucleotide workflow.
2. Add datafile(s) to the Samples Table.
3. Add the oligo nucleobase sequence.
Currently we support a 1-character code. Byos Digested Oligo assumes a phosphodiester backbone, ribose or deoxyribose, and unmodified A, G, C, U, and T. Modifications can be used to change the default sugar, nucleobase, and phosphate linker.
4. Set the mass tolerance settings to meet your instrument's expected accuracy. Use Molecule Type to distinguish RNA and DNA. (NOTE: Before Byos v5.6, Molecule Type was controlled by setting Fragmentation Type to NUVPD for RNA and NETD for DNA).
5. Define RNase Digest Rules.
6a. Set Total Common and Rare Max as needed. See Byonic™ Modification Fine Control for details.
6b. Set the expected 3’ and 5’ Termini to match the RNase used.
If no terminal modifications are applied, Byos Digested Oligonucleotides expects 5’ PO4H2 and 3’ OH.
5’ PO4H2-ACCCAUUG-OH 3’
The Fixed Mods below adjust the Default 3’ and 5’ termini masses for an RNase T1 digested sample.
5’ OH-ACCCAUUG-PO4H2 3’
Hydroxy 5'/ -79.966331 @ NTerm | fixed
Phospho 3' / +79.966331 @ CTerm | fixed
Adding % “comments out” the terminal modification.
5’ OH-ACCCAUUG-OH 3’
Hydroxy 5'/ -79.966331 @ NTerm | fixed
% Phospho 3' / +79.966331 @ CTerm | fixed
6c. Set fixed or variable modifications as needed.
Modifications can be applied to specific bases, or to localized spots with fixed mods on “unused” letters. Any letter excluding A, G, C, T, and U has a default mass value of zero. By applying a fixed modification to an "unused" letter, you can define modified oligo masses that can be localized to specific residues in the full length oligo sequence.
9. Once the workflow is ready, click Create Project. Project investigation tools and automatic report generation are similar to Byos Peptide workflows. The Byos Digested Oligonucleotide Tutorial Video series provides tips and guidance for the Digested Oligonucleotide project investigation view.
You can save the workflow for repeat use.
Optional Steps:
Byos Digested Oligo includes a Wildcard search feature used to discover and localize unknown modifications. A mass range of the modification is defined and the search can be restricted to specific residues.