The Protein Metrics Digested Oligonucletodes workflow is now available for bottom-up sequence confirmation and label-free quantification of digested oligonucleotides.
Structural characterization is essential to guarantee the safety and efficacy of therapeutic mRNA and other large oligonucleotides. LC-MS/MS bottom-up sequence mapping offers a robust solution for assessing and confirming the primary structure of these molecules, as well as detecting modifications. However, this technique encounters several challenges, such as developing the endoribonuclease digestion process, separating oligonucleotide digest products, assigning MS/MS spectra, distinguishing isomers and reporting the data.
The Digested Oligonucleotide workflow is specifically designed for analyzing enzymatically digested oligonucleotides, such as mRNA, using MS/MS acquisitions with high-resolution accurate mass instruments. This workflow utilizes a modified version of the Byonic search engine, which has been repurposed for scoring oligonucleotide spectral matches (OSMs).
This workflow is vendor neutral and can process raw data from all major MS systems.
During project creation, the user can define the expected sequence, set the molecule type (DNA or RNA) and specify the cleavage residue.
Modifications (including user defined) can be incorporated into the MS/MS search:
The inspection view (below) is organised into the following components:
- Project section, lists the raw files in use. Check boxes for MS files and result files can be used to turn on or off the records displayed in the other views. The colors of the files correspond to colors used in the other views.
- Oligonucleotides Coverage displays the percentage sequence coverage for each sample, along with the coverage map
- The table in the center lists all identified Oligonucleotides and can be filtered to show subsets of the results. Selecting a Oligonucleotides row in the table displays the corresponding MS2 (and mass error plot), the extracted ion chromatogram (XIC) and isotope plot.
- The XIC plot displays the ion chromatograms for the selected modified Oligonucleotide and corresponding wildtype*. The XIC boundaries can be adjusted manually if required.
- The MS2 and mass error plots show the MS2 spectrum of the selected Oligonucleotide and corresponding wildtype*.
-The Isotopes plot displays the MS1 isotope profile of the Oligonucleotide and corresponding wildtype*.
*Wildtype comparison must be selected and only shown if the unmodified peptide is present in the data set.
Fig. 3: Digested Oligonucleotides Analysis Dashboard User interface
Coverage map:
Fig. 4: Digested Oligonucleotides GFP sequence coverage map
Bonus tip: If sequences are filtered out of the main table, or samples are deselected in the project section, the coverage will be updated accordingly.
Isomers with distinct mRNA sequences are common for oligonucleotide mapping, which makes confident assignment challenging.
The example below shows a chromatographically separated pair of isomers, which have been automatically assigned in the software, using MS/MS sequence confirmation:
In the example spectra below (A and B), several diagnostic fragment ions are detected, allowing for the unequivocal differentiation between the two isomers.(y2, w2, y4, b2, d2-18, and d4-18).
Here above our table columns show a Delta Score which is the difference in scores between the best (assigned) and the next best assignment.
Effective Oligonucletodies scoring and clear Ion Pairing Reverse Phase chromatography separation are crucial (C).
Our Protein Metrics digested oligonucleotides workflow provides automatic report table displaying numerical oligonucleotides Scores and a heatmap color code illustrating Delta Scores, which is valuable for assessing assignment reliability.