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; Morales-Sanfrutos, Julia; Medina, Rebeca; Lucas, Morghan C.; Novoa, Eva María; Sabidó, Eduard

doi:10.1016/j.chroma.2022.462803

Cited by 10 publications

(12 citation statements)

References 44 publications

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“…The DIA scan window optimization was performed without accounting for retention time effects, making the isolation window scheme applicable for any stationary phase and any chromatographic gradient runtime. This approach differs from a very recently published DIA method by using larger, nonuniform isolation windows designed to account for potential isobaric fragment ions . Compared to small, uniform isolation windows, this allows for greater points across a peak and/or higher AGC targeting, enhancing quantitative precision and/or sensitivity.…”

Section: Resultsmentioning

confidence: 99%

“…This approach differs from a very recently published DIA method by using larger, nonuniform isolation windows designed to account for potential isobaric fragment ions. 23 Compared to small, uniform isolation windows, this allows for greater points across a peak and/or higher AGC targeting, enhancing quantitative precision and/or sensitivity.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Data-Independent Acquisition for the Detection of Mononucleoside RNA Modifications by Mass Spectrometry

Janssen

Xie

Kramer

et al. 2022

J. Am. Soc. Mass Spectrom.

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RNA is dynamically modified in cells by a plethora of chemical moieties to modulate molecular functions and processes. Over 140 modifications have been identified across species and RNA types, with the highest density and diversity of modifications found in tRNA (tRNA). The methods used to identify and quantify these modifications have developed over recent years and continue to advance, primarily in the fields of next-generation sequencing (NGS) and mass spectrometry (MS). Most current NGS methods are limited to antibody-recognized or chemically derivatized modifications and have limitations in identifying multiple modifications simultaneously. Mass spectrometry can overcome both of these issues, accurately identifying a large number of modifications in a single run. Here, we present advances in MS data acquisition for the purpose of RNA modification identification and quantitation. Using this approach, we identified multiple tRNA wobble position modifications in Arabidopsis thaliana that are upregulated in salt-stressed growth conditions and may stabilize translation of salt stress induced proteins. This work presents improvements in methods for studying RNA modifications and introduces a possible regulatory role of wobble position modifications in A. thaliana translation.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

Data-Independent Acquisition for the Detection of Mononucleoside RNA Modifications by Mass Spectrometry

Janssen

Xie

Kramer

et al. 2022

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

show abstract

“…Despite the pivotal function that tRNAs play in cellular processes and their involvement in numerous human diseases, we currently lack a simple and cost-effective method to accurately quantify both tRNA abundances and their modifications systematically. On the one hand, tRNA modifications are typically identified and quantified with high accuracy using liquid chromatography coupled to mass spectrometry (LC-MS) methodologies 48,[64][65][66][67][68][69] . In these methods, RNA molecules are fragmented into oligomers or monomers, and their abundance is measured via UV absorption or MS/MS.…”

Section: Extending Trna Ends With Rna Adapters Improves Basecallingmentioning

confidence: 99%

Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing

et al. 2023

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Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment. We show that default nanopore sequencing settings discard the vast majority of tRNA reads, leading to poor sequencing yields and biased representations of tRNA abundances based on their transcript length. Re-processing of raw nanopore current intensity signals leads to a 12-fold increase in the number of recovered tRNA reads and enables recapitulation of accurate tRNA abundances. We then apply Nano-tRNAseq to Saccharomyces cerevisiae tRNA populations, revealing crosstalks and interdependencies between different tRNA modification types within the same molecule and changes in tRNA populations in response to oxidative stress.

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“…Thus far, various HILIC stationary phases, including pure silica gel, and chemically bonded silica phases with a diol, amide, amino, or zwitterionic, have been commercialized and widely used [2]. Increasing demand for the analysis of highly polar compounds in biological samples (such as metabolites) has fueled research on developing miniaturized column technologies, which are amenable to the analysis of a minute sample with high performance and fast separation [3–6]. Nevertheless, packing the HPLC packing materials into the fused silica capillary requires tremendous skills.…”

Section: Introductionmentioning

confidence: 99%

Preparation of poly(N‐vinylpyrrolidone‐co‐pentaerythritol triacrylate) monolithic column for hydrophilic interaction chromatography

Qiu

et al. 2023

J of Separation Science

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A method for the preparation of poly(N-vinylpyrrolidone-co-pentaerythritol triacrylate copolymerization)-based monolithic capillary column was reported for the separation of polar small molecular weight compounds with nano-liquid chromatography in hydrophilic interaction chromatography mode. The monolithic columns were prepared by in situ copolymerization of N-vinylpyrrolidone and a cross-linker pentaerythritol triacrylate in a binary porogenic agent consisting of methanol and water. The composition of the polymerization solution was systematically optimized in terms of column permeability, theoretical plate number, asymmetric factor, and retention factor. A typical hydrophilic chromatography retention mechanism was observed with a mobile phase composed of a high content of organic solvent. The preparation method is simple and robust, the precursor N-vinylpyrrolidone is chemically stable, cheap, and easily available. The N-vinylpyrrolidone-based hydrophilic interaction chromatography stationary phase displays satisfactory separation selectivity for a range of polar test analytes, including benzoic acid derivatives, nucleosides, and phenols.

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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

Cited by 10 publications

References 44 publications

Data-Independent Acquisition for the Detection of Mononucleoside RNA Modifications by Mass Spectrometry

Data-Independent Acquisition for the Detection of Mononucleoside RNA Modifications by Mass Spectrometry

Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing

Preparation of poly(N‐vinylpyrrolidone‐co‐pentaerythritol triacrylate) monolithic column for hydrophilic interaction chromatography

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