title: Self reporting RNA probes as an alternative to cleavable small molecule mass tags.
abstract: The large size of biological molecules such as proteins and oligonucleotides makes them inherently problematic to analyse and quantify directly by mass spectrometry. For these molecules, electrospray ionisation produces multiply charged species and associated alkali metal adducts which can reduce sensitivity and complicate quantification. Whereas time-of-flight mass analysers, often coupled to matrix-assisted laser desorption/ionisation, can have insufficient mass resolution to resolve these large molecules in the higher m/z range. This has led to the development of cleavable small molecule mass tag approaches for the indirect analysis of biomolecules such as proteins and oligonucleotides. Existing methodologies require the design and synthesis of a cleavable linker to join the biomolecule and the mass tag. Here, an alternative approach to small molecule mass tags is presented, which exploits the properties of the RNA molecule to afford self-reporting probes which can be easily synthesised using automated phosphoramidite chemistry. The sugar-phosphate backbone of RNA was used as a built-in enzyme cleavable linker and through the use of RNase digestion of bromine labelled oligonucleotides the observation of a range of small molecule mass tags by mass spectrometry is demonstrated. This study provides a proof-of-concept that RNase digestion can be used to produce labelled small molecule mass tags from oligonucleotide probes, thus eliminating the need for custom design and synthesis of a cleavable linker.
authors.affiliation: Chemistry, FNES, University of Southampton, Southampton, SO17 1BJ, UK.
authors.forename: Jo-Anne
authors.initials: JA
authors.lastname: Riley
authors.forename: Tom
authors.initials: T
authors.lastname: Brown
authors.forename: Nittaya
authors.initials: N
authors.lastname: Gale
authors.forename: Julie
authors.initials: J
authors.lastname: Herniman
authors.forename: G John
authors.initials: GJ
authors.lastname: Langley
mesh_terms: D000577:Amides / Q000737:chemistry; D001483:Base Sequence; D013058:Mass Spectrometry / Q000379:methods*; D010756:Phosphoric Acids / Q000737:chemistry; D015347:RNA Probes / Q000737:chemistry* / Q000235:genetics
chemical_list: D000577:Amides; D010756:Phosphoric Acids; D015347:RNA Probes; C011067:phosphoramidic acid