Dataset Information

High-performance nano-flow liquid chromatography column combined with high- and low-collision energy data-independent acquisition enables targeted and discovery identification of modified ribonucleotides by mass spectrometry (Mixed-mode column chromatography)

ABSTRACT:

Over 170 post-transcriptional RNA modifications have been described and are common in all kingdoms of life. These modifications range from methylation to complex chemical structures, with methylation being the most abundant. RNA modifications play a key role in RNA folding and function and their dysregulation in humans has been linked to several diseases such as cancer, metabolic diseases or neurological disorder. Nowadays, liquid chromatography-tandem mass spectrometry is considered the gold standard method for the identification and quantification of these modifications due to its sensitivity and accuracy. However, the analysis of modified ribonucleosides by mass spectrometry is complex due to the presence of positional isomers. In this scenario, optimal separation of these compounds by highly sensitive liquid chromatography combined with the generation of high-information spectra is critical to unequivocally identify them, especially in high-complex mixtures. Here we present an analytical method that comprises a new type of mixed-mode nano-flow liquid chromatography column combined with high- and low-collision energy data-independent mass spectrometric acquisition for the identification and quantitation of modified ribonucleosides. The method produces content-rich spectra and combines targeted and screening capabilities thus enabling the identification of a variety of modified nucleosides in biological matrices by single-shot liquid chromatographic analysis coupled to mass spectrometry.

Mixed-mode column chromatography is reported in the current study MTBLS2497.

Data-independent acquisition is reported in MTBLS2959.

INSTRUMENT(S): Liquid Chromatography MS - positive - reverse phase

SUBMITTER: guadalupe espadas

PROVIDER: MTBLS2497 | MetaboLights | 2023-10-26

REPOSITORIES: MetaboLights

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Dataset's files

Source:

			Action	DRS
	MTBLS2497	Other
	FILES	Other
	a_MTBLS2497_LC-MS_positive_reverse-phase_metabolite_profiling.txt	Txt
	i_Investigation.txt	Txt
	m_MTBLS2497_LC-MS_positive_reverse-phase_metabolite_profiling_v2_maf.tsv	Tabular

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Publications

High-performance nano-flow liquid chromatography column combined with high- and low-collision energy data-independent acquisition enables targeted and discovery identification of modified ribonucleotides by mass spectrometry.

Espadas Guadalupe G Morales-Sanfrutos Julia J Medina Rebeca R Lucas Morghan C MC Novoa Eva Maria EM Sabidó Eduard E

Journal of chromatography. A 20220102

PMID: 35042139

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Project description:N6-methyladenosine (m6A) is the methylation of adenosine at position N6 and is notably one of the most abundant modifications found in mRNA of eukaryotes. Although it does not interrupt the coding functions of transcripts, it assists in regulating several RNA functions like splicing, stability, translocation and translation and is associated with disease development, cell proliferation, cell migration, cell invasion, autophagy, cell apoptosis, and drug resistance. In this study, we performed a colorimetric assay, ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and methylated RNA immunoprecipitation sequencing (MeRIP-seq) to assess the RNA N6-methyladenosine landscape of Echinococcus granulosus; a tapeworm species of the phylum Platyhelminthes that is responsible for a cosmopolitan zoonosis (cystic echinococcosis) of public health importance. While the levels of m6A modification were not significantly different between the life cycle stages, many important genes including notably enriched and expanded protein domains like Dyneins, Hsp70, and Cadherin thought to play important roles in adaptation to parasitism were m6A-modified. These genes were functionally enriched in biological processes like cell division, cell adhesion, microtubule-based movement, important pathways involved in parasitism (e.g. MAPK), and disease processes. Additionally, the m6A alterations were present in the first exon of modified genes, suggesting gene regulatory activities. Conclusively, m6A modification of the mRNA is present in Echinococcus, and is mediated by orthologues of known mammalian methyltransferases but possibly irreversible due to the absence of demethylases. The demonstration of differentially m6A-methylation in the different life cycle stages of Echinococcus suggests a role in the development and survival of the tapeworm. It also provides the groundwork for future studies in understanding the implication of RNA methylation in the treatment of echinococcoses.