Introduction
Oligonucleotides have been rapidly gaining attention, as these therapeutic modalities hold high promise to previously untreatable or rare diseases and can be better alternatives for common diseases. Oligonucleotide based pharmaceuticals are produced by chemical synthesis and that often results in various product-related impurities, including deletion sequences (‘shortmers’), addition sequences (‘longmers’), and modifications. Oligonucleotide characterization is necessary to ensure safety and efficacy of therapeutic oligonucleotides.
The Oligo module allows users to evaluate a variety of nucleotide macromolecules such as: DNA, RNA, and custom assemblies, including DNA/RNA combinations, phosphorothioate backbones, and user specified modifications. This application note details how to use the Byos Oligo Workflow for oligonucleotides analysis, validation, quantification, and reporting.
Quick Guide to Executing the Byos Oligo Workflow
- Click Oligo to launch the workflow
- Drag & Drop raw data files
- Drag & Drop a FASTA file or create using “Building Blocks”
- Define tolerances, delta masses, and clips
- Set deconvolution parameters
- Create a project
Configuring the Workflow
Sample Input
The Oligonucleotide module supports MS1 only, mixed MS1 and MS2, and targeted MS2 based methods. Byos automatically annotates matching peaks pulling MS2 data according to the user-defined MS1/MS2 associations. Users can associate one or more separate MS2 data files to MS1 data to allow for targeted MS2 based methods.
- MS1 only and mixed MS1 / MS2 Set-Up
To add a file, drag and drop an MS raw file into the project window. Alternatively, click Add sample, double-click in the MS file column, click the icon and browse to the sample file.
- Targeted MS2 Analysis Set-Up
To associate multiple MS2 datafiles to a single sample, first add or drag and drop the MS file into the MS file column. Then double-click in the MS/MS file column and select the icon to add the targeted MS2 file. Select the
icon to associate additional targeted MS2 files. The below figure shows 5 different targeted MS2 files associated to one MS file.
Sequence Input
The user can add a nucleotide sequence to the Chains table by either dragging and dropping a FASTA file, clicking Select from FASTA file, or adding the sequence manually. To manually enter the sequence, click Add in the Chains section, and click in the Name and Sequence/average mass cells to enter text in those columns. The Building blocks sub-tab contains information on naming convention for building custom oligonucleotide sequences. The user has an option to add new building blocks or edit the existing building blocks to change chemical formula or the mass. For more information on Building Blocks please refer to Oligonucleotide Building Blocks Basics.
The chains can then be added as rows to the Sequence combinations table by selecting Mirror Chains table checkbox. The user can edit the Sequence combinations table only after unselecting Mirror Chains table checkbox. Byos automatically computes the average and monoisotopic mass for each sequence combination entry, and these masses are then available for automatic peak assignment.
Mass Matching – Tolerances, Delta Masses, and Clips
The Tolerances sub-tab allows for setting the precursor mass tolerance (MS1) and the fragment mass tolerance (MS2). Users can select either Average or Monoisotopic mass match option for the MS1 tolerance. Monoisotopic mass matching is ideal for smaller molecules when resolution is sufficiently high.
The Delta masses sub-tab gives a table of likely mass differences between observed peaks and the reference mass input by the user or computed from an oligonucleotide sequence. The table includes delta masses of common oligonucleotide impurities.
In addition, clips and truncations can be anticipated from either 5' or 3' termini or both. This feature can be enabled by checking Consider Oligo impurities and setting the parameters in the Maximum clipped residues box.
Note: The users can then go to the Masses table within the project and check the Oligo Candidate column to see the annotation of the internal clips.
The Single end column limits the maximum number of residues clipped from either the 5' or 3' terminal. The Both ends column limits the number of clipped residues from either the 5' or 3' terminal when the opposite terminal is also clipped. The below example illustrates single clipping from either the 5' or 3' prime end and clipping from both the 5' and 3' prime ends.
Spectrum Processing
Users can specify the mass range, m/z range, minimum difference between mass peaks, and maximum number of mass peaks applicable for each project. The Advanced sub-tab mainly concerns resolution, and these parameters are only needed if analyzing larger species. Please contact Protein Metrics support for additional information.
Processing Node
A set of advanced commands specific for oligonucleotide analysis is incorporated into the default workflow Processing Node tab. Please refer to the Oligonucleotide Analysis: Advanced Commands section of this user guide for more detailed information on these advanced commands.
Inspecting Results
Once deconvolution and mass matching are complete, a project will be created to validate identified oligonucleotide candidates and produce a comprehensive report. The inspection view includes a trace plot, trace peaks table, masses table, MS1 spectrum and deconvolution mass spectrum. A MS2 data table and MS2 spectrum will be available for projects that contain MS2 information.
- Trace Peak Table: Select an integrated trace peak in the Trace Peaks table to investigate the oligonucleotide candidates identified.
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Masses Table: Select an oligonucleotide candidate or mass in the Masses Table to update the MS1 and deconvoluted plot spectrum. When a mass(es) is selected, the mass to m/z dot rendering will link neutral mass peaks to the MS1 spectrum and allow the user to investigate the results. Multiple deconvoluted masses can be selected by using the
icon and dragging the box over peaks of interest or by selecting multiple masses in the Masses table. Orange diamonds will denote the monoisotopic mass.
- MS2 Data Table and MS2 Plot: The MS2 Data table will be populated when an oligonucleotide candidate or mass which contains MS2 data is selected. Each row in the table represents a MS2 scan of a single charge state of the selected oligonucleotide candidate or mass. The MS2 Data table provides information of the sequences, along with other details such as the mass, charge state, acquisition time, and score. The score is a value which reflects the probability of randomly matching observed peaks to theoretical fragments. Selecting a MS2 row will populate the MS2 Plot with the corresponding fragments and m/z error plot. The user can sum MS2 scans by selecting the check box in the Selected column. Additional MS2 information can allow the user to interpret fragment ions and allow nucleotide sequence verification.
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Mass XIC: The Mass XIC feature allows the user to track the elution profiles of co-eluting masses in complex matrixes and identify in-source species. To enable select Window -> Enable Mass XIC.
Creating Reports
The Oligo report template is automatically loaded for Byos-created projects. If not, the user can add the report by selecting the report icon located below the Help menu. Click “Export ->Export to PDF/Excel Workbook…” to generate a single pdf or excel file with all the tabs. For more information on how to customize this report, please see our related videos or contact us.
The report configuration includes:
- A summary tab with details of the Project Status (Traffic Light view). The traffic light validations can be edited in the investigative view within the R project table. For more information see the Traffic Light (Pass/Fail) View in Intact Workflows article.
- Pivot tables to tabulate intensities for both Expected and All Masses.
- Pivot tables to tabulate relative intensity % for both Expected and All Masses. Each vertical column will add up to 100% and allow the user to better understand which oligonucleotide fragments predominate each sample.
- A bar chart comparison of the relative intensity of oligonucleotide candidates.
- Pivot table to tabulate mass error (ppm) between expected and observed masses.
- A plot tab displaying the whole trace plot and the individual integrated trace peaks with the corresponding MS1 and deconvoluted mass spectra.
- A project options tab that details the workflow processing parameters.