Poster number 131 submitted by Ila Marathe
Establishing robust calibration curves for mass photometry-based measurements 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)
Mass photometry (MP) measures masses of biomolecules using interferometric scattering microscopy1. This method can be used to rapidly assess sample heterogeneity, composition, and macromolecular interactions. Typically, molecules with known masses and similar optical properties as the analytes (e.g., proteins, nucleic acids) are used for calibration. However, calibrations generated with either RNA or protein may lead to incorrect mass assignments for ribonucleoprotein (RNP) complexes2. We sought to develop alternatives that would allow greater accuracy during MP studies of RNPs; mass measurements were independently validated by high-resolution native mass spectrometry (nMS). Preliminary studies were conducted using archaeal RNase P, an essential, multi-subunit, catalytic RNP3-5, consisting of one RNase P RNA (RPR) and five RNase P proteins (RPPs). RPR can be reconstituted with different suites of RPPs, to form RNPs of varying RNA:protein ratios. First, protein- and RNA-only calibrations were generated from the ratiometric contrasts obtained by MP-based measurements of biomolecules with known molecular masses. Beta-amylase and thyroglobulin (present as multimeric complexes) were used for protein calibration. In vitro transcribed RNAs of known sizes (including some larger RNAs generated by RNA engineering) were used for RNA-based calibrations. Masses of protein and RNA standards were measured with MP using appropriate calibrations and were found to be in good agreement (within 0 – 6%) of expected and nMS-generated masses. To evaluate the suitability and appropriateness of RNA and protein calibrations in estimating masses of RNP complexes, RPR was reconstituted with either two or five RPPs (67% and 49% RNA content respectively). Despite significant fractional mass contribution of the RPR, protein calibration provided more accurate mass measurements (0.5 – 2.6% of expected mass) compared to the RNA calibration (5 – 7% of expected mass). Studies with a more structurally diverse set of RNPs, followed by mass validation using nMS, are needed to establish optimal mass calibration protocols for RNPs.
1. Young G and Kukura P. (2019), Annu. Rev. Phys. Chem., 70: 301-322.
2. Lai SH et al. (2021), iScience, 24: 103211.
3. Zahurancik WJ et al (2021), Meth. Enzymol., 649: 71-103.
4. Marathe IA et al (2021), Nucleic Acids Res., 49: 9444-9458.
5. Phan HD et al. (2022), Nucleic Acids Res, 50: 8154-8167.
Keywords: Mass photometry, Native mass spectrometry, Ribonucleoproteins