MIND4OLIGOS: Determining the Monoisotopic Mass of Oligonucleotides Observed in High-Resolution Mass Spectrometry

Abstract

Oligonucleotide therapeutics have emerged as an important class of drugs offering targeted therapeutic strategies that complement traditional modalities, such as monoclonal antibodies and small molecules. Their unique ability to precisely modulate gene expression makes them vital for addressing previously undruggable targets. A critical aspect of developing these therapies is characterizing their molecular composition accurately. This includes determining the monoisotopic mass of oligonucleotides, which is essential for identifying impurities, degradants, and modifications that can affect the drug efficacy and safety. Mass spectrometry (MS) plays a pivotal role in this process, yet the accurate interpretation of complex mass spectra remains challenging, especially for large molecules, where the monoisotopic peak is often undetectable. To address this issue, we have adapted the MIND algorithm, originally developed for top-down proteomics, for use with oligonucleotide data. This adaptation allows for the prediction of monoisotopic mass from the more readily detectable, most-abundant peak mass, enhancing the ability to annotate complex spectra of oligonucleotides. Our comprehensive validation of this modified algorithm on both in silico and real-world oligonucleotide data sets has demonstrated its effectiveness and reliability. To facilitate wider adoption of this advanced analytical technique, we have encapsulated the enhanced MIND algorithm in a user-friendly Shiny application. This online platform simplifies the process of annotating complex oligonucleotide spectra, making advanced mass spectrometry analysis accessible to researchers and drug developers. The application is available at https:// valkenborg-lab.shinyapps.io/mind4oligos/.

Nucleo-SAFARI: Automated Identification of Fragment Ions in Top-Down MS/MS Spectra of Nucleic Acids

“…50 Finally, MIND4OLIGOS recently introduced an algorithm for rapid determination of oligonucleotide monoisotopic mass. 51 Each of these tools has offered substantial progress in the field of RNA MS, leveraging its strengths to characterize biologically relevant molecules. Each of these software methods, however, focuses on the interrogation of LC-MS/MS data of small (typically <30 nt) RNAs produced by digestion with restriction enzymes, limiting their applicability to top-down analyses of nucleic acids.…”