Poster abstracts
Poster number 31 submitted by Ila Marathe
Use of native mass spectrometry and mass photometry for mass characterization of RNAs and ribonucleoprotein complexes
Ila A. Marathe (Department of Chemistry and Biochemistry; Resource for Native Mass Spectrometry-Guided Structural Biology; Center for RNA Biology; The Ohio State University), Stella M. Lai (Department of Chemistry and Biochemistry; Resource for Native Mass Spectrometry-Guided Structural Biology; Center for RNA Biology; The Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry; Center for RNA Biology; The Ohio State University), Vicki H. Wysocki (Department of Chemistry and Biochemistry; Resource for Native Mass Spectrometry-Guided Structural Biology; Center for RNA Biology; The Ohio State University)
Abstract:
The significance of RNA therapeutics emphasizes the need for rapid quality control and structural characterization of RNAs and ribonucleoproteins (RNPs). Native mass spectrometry (nMS), which retains the native structure and stoichiometry of large biomolecules in the gas phase, can measure accurate masses of large RNAs. Here, nMS was used to gauge the ability of published methods to minimize the 3' heterogeneity of RNAs transcribed in vitro by T7 RNA polymerase (RNAP). Transcript analysis using Thermo Scientific Q Exactive Ultra High Mass Range mass spectrometer revealed a mixture of expected length (n) and longer (e.g., n+1, n+2) RNAs. To minimize 3' heterogeneity due to untemplated nucleotide addition, we transcribed RNAs using (a) modified DNA templates, (b) capture oligonucleotides to prevent transcript self-priming, or (c) a thermostable T7 RNAP. We also site-specifically cleaved some transcripts by RNase H. nMS analysis allowed subsequent comparison of efficacy of these approaches, demonstrating the benefits of using nMS to rapidly study large RNAs. However, while nMS is mostly suitable for studying RNAs/RNPs, volatile electrolytes needed for ionization may be incompatible with some analytes. Therefore, we used mass photometry (MP), a light scattering-based technique, for solution-phase studies. MP uses mass calibrants with similar optical properties as the analyte (e.g., proteins, nucleic acids) to measure masses. Since analyte optical properties dictate calibrant choice, using a single calibrant can introduce ambiguity into mass analyses of complex molecules like RNPs. Using RNase P RNP assemblies with varying RNA–protein content as test samples and either RNA- or protein-based calibrations, we sought to establish an appropriate MP calibrant reference for accurate mass measurements of RNPs. While RNA calibration provided a more accurate mass (0–0.72% of expected) for an RNP with 88% RNA, protein calibration provided more accurate masses (0.5–2.6% of expected) for RNPs with 67% or 49% RNA than the RNA calibration did (5–7% of expected). Our data suggest that RNP composition and solvent accessibility dictate calibrant choice. Mass validation by nMS is ongoing. These studies are expected to provide a framework for characterizing RNAs and RNPs through the integrated use of nMS and MP.
Keywords: Native mass spectrometry, Mass photometry, RNAs and RNPs