Normalized retention time for scheduled liquid chromatography-multistage mass spectrometry analysis of epitranscriptomic modifications.
J Chromatogr A
; 1623: 461181, 2020 Jul 19.
Article
em En
| MEDLINE
| ID: mdl-32505282
ABSTRACT
Investigations into post-transcriptional modifications of RNA and their regulatory proteins have revealed pivotal roles of these modifications in cellular functions. A robust method for the quantitative analysis of modified nucleosides in RNA may facilitate the assessment about their functions in RNA biology and disease etiology. Here, we developed a sensitive nano-liquid chromatography-multistage mass spectrometry (nLC-MS3) method for profiling simultaneously 27 modified ribonucleosides. We employed normalized retention time (iRT) and scheduled selected-reaction monitoring (SRM) to achieve high-throughput analysis, where we assigned iRT values for modified ribonucleosides based on their relative elution times with respect to the four canonical ribonucleosides. The iRT scores allowed for reliable predictions of retention times for modified ribonucleosides with the use of two types of stationary phase materials and various mobile phase gradients. The method enabled the identification of 20 modified ribonucleosides with the use of the enzymatic digestion mixture of 2.5 ng total RNA and facilitated robust quantification of modified cytidine derivatives in total RNA. Together, we established a scheduled SRM-based method for high-throughput analysis of modified ribonucleosides with the use of a few nanograms of RNA.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Massas
/
Cromatografia Líquida
/
Epigênese Genética
/
Transcriptoma
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
J Chromatogr A
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
Estados Unidos