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 (Data-independent acquisition)

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.

Data-independent acquisition is reported in the current study MTBLS2959.

Mixed-mode column chromatography is reported in MTBLS2497.

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

SUBMITTER: guadalupe espadas

PROVIDER: MTBLS2959 | MetaboLights | 2023-10-26

REPOSITORIES: MetaboLights

ACCESS DATA

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

Similar Datasets

Project description:This series represents a group of five samples (from sigmoid colon biopsies of clinically normal Patients A-E) profiled by a publicly available cDNA-based platform (GPL284) and a commercial oligonucleotide-based platform (GPL91). Sample RNA extraction: Biopsies taken during endoscopic analysis were immediately snap-frozen in liquid nitrogen and at no time prior to or during subsequent sample manipulation were the samples allowed to thaw. Standard laboratory procedures to eliminate the presence of RNases, including the use of RNase free plastic-ware, heat baked glassware, molecular biology grade chemicals and DEPC-treated solutions, were adopted. Frozen biopsies were crushed to a fine powder under liquid nitrogen using a manual crusher with a teflon head (Omnilab) and total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturers protocol, with minor modifications that improved RNA yields and purity in our hands. In brief, crushed biopsy samples were lysed in an appropriate volume of Buffer RLT (typically 600 µL/20 mg tissue). To facilitate complete lysis, samples were allowed to stand at room temperature for 5-10 min before vortexing. The lysate was homogenized by addition to a QIA shredder column (Qiagen) and centrifuged at 14,000 rpm for 2 min. The resulting supernatant was centrifuged at 14,000 rpm for 3 min to pellet any cell debris. One volume of 70% ethanol was added to the cleared lysate, added to the RNeasy column and centrifuged at 10,000 rpm for 15 sec. Bound RNA was washed with the addition of RW1 buffer to the column and allowed to stand at room temperature for 10 min followed by centrifugation at 10,000 rpm for 15 sec. DNase 1 incubation mix (Stock: 273 kunits; RNase-free DNase Set, Qiagen) was added directly to the spin column membrane and digestion of any contaminating genomic DNA was carried out at room temperature for 30 min. The column was washed with RWI buffer and the DNase treatment step was repeated. The bound RNA was washed with RW1 buffer and centrifuged at 10,000 rpm for 15 sec three times, followed by two additional wash and centrifugation steps with RPE buffer. Finally, purified RNA was eluted off the column with RNase-free water. RNA concentration and purity was determined spectrophotometrically at A260/A280 and was determined to be intact as assessed by formaldehyde gel electrophoresis. The presence of genomic DNA contamination was assessed by PCR amplification of GAPDH using standard PCR conditions and the following primers: GAPDH_F2-5’- ACCCACTCCTCCACCTTTGAC-3’, GAPDH_R2- 5’-CTGTTGCTGTAGCCAAATTCGT-3’. RNA was re-treated with DNase if necessary. Upon confirming the quality of the RNA (intact and free of genomic DNA), 15 µg of total RNA from each of the five biological replicates was used for expression screening on either the Affymetrix or the clone-based filter platform. Keywords: parallel sample

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 (Data-independent acquisition)

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.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure