Absolute and relative quantification of RNA modifications via biosynthetic isotopomers
Nucleic acids research. Bd. 42. H. 18. Oxford: Oxford Univ. Press 2014 e142
Erscheinungsjahr: 2014
ISBN/ISSN: 1362-4962 ; 0305-1048
Publikationstyp: Zeitschriftenaufsatz
Sprache: Englisch
Doi/URN: 10.1093/nar/gku733
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Inhaltszusammenfassung
In the resurging field of RNA modifications, quantification is a bottleneck blocking many exciting avenues. With currently over 150 known nucleoside alterations, detection and quantification methods must encompass multiple modifications for a comprehensive profile. LCMS/MS approaches offer a perspective for comprehensive parallel quantification of all the various modifications found in total RNA of a given organism. By feeding 13 C-glucose as sole carbon source, we have generated a stable is...In the resurging field of RNA modifications, quantification is a bottleneck blocking many exciting avenues. With currently over 150 known nucleoside alterations, detection and quantification methods must encompass multiple modifications for a comprehensive profile. LCMS/MS approaches offer a perspective for comprehensive parallel quantification of all the various modifications found in total RNA of a given organism. By feeding 13 C-glucose as sole carbon source, we have generated a stable isotope-labeled internal standard (SIL-IS) for bacterial RNA, which facilitates relative comparison of all modifications. While conventional SIL-IS approaches require the chemical synthesis of single modifications in weighable quantities, this SIL-IS consists of a nucleoside mixture covering all detectable RNA modifications of Escherichia coli, yet in small and initially unknown quantities. For absolute in addition to relative quantification, those quantities were determined by a combination of external calibration and sample spiking of the biosynthetic SIL-IS. For each nucleoside, we thus obtained a very robust relative response factor, which permits direct conversion of the MS signal to absolute amounts of substance. The application of the validated SIL-IS allowed highly precise quantification with standard deviations » weiterlesen» einklappen
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DDC Sachgruppe:
Naturwissenschaften