PXD070755

PXD070755 is an original dataset announced via ProteomeXchange.

Title	Comprehensive Evaluation of RNases, Nano-Flow HILIC Chromatography, and Mass Spectrometry for Shotgun RNomics of Native RNA
Description	RNA modifications play crucial roles in regulating cellular processes, but comprehensive mapping of the human RNome still remains limited by technological challenges. Mass spectrometry is a valuable tool to analyse RNA modifications and complementing sequencing-based analysis. Current MS-based oligonucleotide workflows have limited sensitivity, requiring micrograms of RNA inputs and thus hindering studies on native RNAs. Additionally, environmentally toxic ion-pairing reagents are often required. There is also a demand for more specific nucleases and systematic studies on different nucleases. Here, we report a highly sensitive, broadly applicable oligonucleotide-MS workflow that enables analysis of nanogram-scale RNA hydrolysates. We systematically benchmarked the substrate specificity of three nucleases: RNase T1, RNase 4, and Colicin E5 under varying conditions. We developed a nano-flow hydrophilic interaction liquid chromatography (HILIC) setup compatible with common MS buffers and coupled this with high-resolution MS. Using modified NucleicAcidSearchEngine (NASE), we confidently assigned RNA hydrolysates with diverse 3’-end chemistries. Furthermore, we demonstrate that RNase 4 and Colicin E5 efficiently cleave modified RNAs including pseudouridine-containing transcripts, enabling high sequence coverages. Using this workflow, we successfully mapped modifications in 25 ng of native yeast tRNAPhe and verified the sequence of 250 ng of synthetic mRNA. Overall, our method provides a sensitive, high-resolution platform for shotgun RNomics, facilitating comprehensive analysis of RNA modifications in limited biological samples and advancing efforts toward complete epitranscriptomic mapping.
HostingRepository	PRIDE
AnnounceDate	2026-09-07
AnnouncementXML	Submission_2026-09-07_04:25:36.384.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Jannick Schicktanz
SpeciesList	scientific name: Escherichia coli; NCBI TaxID: NEWT:562;
ModificationList	No PTMs are included in the dataset
Instrument	ultraflex; Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2025-11-14 12:29:59	ID requested
⏵ 1	2026-09-07 04:25:37	announced

10.1093/nar/gkag691;

Qi Y, Li C, Yesiltac-Tosun N, Schicktanz J, Rusling L, Kaiser S, Wein S, Kaiser S, A nanogram-sensitive workflow for oligonucleotide mass spectrometry using ion-pair-free nanoflow HILIC and RNase benchmarking. Nucleic Acids Res, 54(13):(2026) [pubmed]

submitter keyword: RNase, RNome, oligonucleotide MS,HILIC, nano-flow, LC-MS/MS

Stefanie Kaiser
contact affiliation	Kaiser Isotope Lab, Department of Pharmaceutical Chemistry, Goethe University Frankfurt, Frankfurt 60438
contact email	stefanie.kaiser@pharmchem.uni-frankfurt.de
lab head
Jannick Schicktanz
contact affiliation	Goethe University Frankfurt am Main
contact email	jschickt@em.uni-frankfurt.de
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD070755

PRIDE project URI

• PRIDE
  1. PXD070755
     1. Label: PRIDE project
     2. Name: Comprehensive Evaluation of RNases, Nano-Flow HILIC Chromatography, and Mass Spectrometry for Shotgun RNomics of Native RNA