PXD033334-1

PXD033334 is an original dataset announced via ProteomeXchange.

Title	Human RNase 4 Improves mRNA Sequence Characterization by LC-MS/MS.
Description	With the rapid growth of synthetic messenger RNA (mRNA)-based therapeutics and vaccines, the development of analytical tools for characterization of long, complex RNAs has become essential. Tandem liquid chromatography-mass spectrometry (LC-MS/MS) permits direct assessment of the mRNA primary sequence and modifications thereof without conversion to cDNA or amplification. It relies upon digestion of mRNA with site-specific endoribonucleases to generate pools of short oligonucleotides that are then amenable to MS-based sequence analysis. Here, we showed that the uridine-specific human endoribonuclease hRNase 4 improves mRNA sequence coverage, in comparison with the benchmark enzyme RNase T1, by producing a large population of uniquely mappable cleavage products. Furthermore, we deployed hRNase 4 to characterize mRNAs fully substituted with 1-methylpseudouridine (m1Ψ) or 5-methoxyuridine (mo5U), as well as mRNAs selectively depleted of uridine–two key strategies to reduce synthetic mRNA immunogenicity. Lastly, we demonstrated that hRNase 4 enables direct assessment of the 5′ cap incorporation in in vitro synthesized mRNA. Collectively, this study highlights the power of hRNase 4 to interrogate mRNA sequence, identity, and modifications by LC-MS/MS.
HostingRepository	PRIDE
AnnounceDate	2022-07-26
AnnouncementXML	Submission_2022-07-26_11:08:26.935.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Eric Wolf
SpeciesList	scientific name: synthetic RNA; NCBI TaxID: 2086595;
ModificationList	methylated residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2022-04-21 05:55:42	ID requested
⏵ 1	2022-07-26 11:08:27	announced
2	2023-11-14 08:55:39	announced	2023-11-14: Updated project metadata.

10.1093/NAR/GKAC632;

submitter keyword: mRNA

Human RNase4;

Ivan R. Correa Jr.
contact affiliation	New England Biolabs, Inc, 240 County Rd, Ipswich, MA, 01938, USA.
contact email	correa@neb.com
lab head
Eric Wolf
contact affiliation	New England Biolabs
contact email	ewolf@neb.com
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/2022/07/PXD033334

PRIDE project URI

• PRIDE
  1. PXD033334
     1. Label: PRIDE project
     2. Name: Human RNase 4 Improves mRNA Sequence Characterization by LC-MS/MS.