PXD073683

PXD073683 is an original dataset announced via ProteomeXchange.

Title	Rapid Histone Post-Translational Modification Analysis Using Alternative Proteases and Tandem Mass Tags (RIPUP)
Description	Histone post-translational modifications (PTMs) alter chromatin dynamics and contribute to the regulation of gene expression in health and disease. Mass spectrometry-based analysis is the gold-standard for histone PTM analysis, but it remains constrained by inefficient sample preparation workflows requiring multiple days. Here, we develop RIPUP (Rapid Identification of histone PTMs in Underivatized Peptides), a streamlined multi-protease workflow that reduces sample preparation from days to hours while improving PTM coverage and quantitative accuracy. Through systematic evaluation of Arg-C Ultra and a prototype recombinant (r)-Chymotrypsin proteases from Promega™ under varied conditions, with or without chemical derivatization using propionic anhydride and tandem mass tags (TMT), we demonstrate that Arg-C Ultra with TMT labeling achieves comparable total PTM detection to conventional Trypsin-based approaches. Using the HiP-Frag computational framework for unrestrictive PTM identification, we discover that TMT's tertiary amine provides charge compensation that rescues ionization of negatively charged acylation marks, revealing 50 succinylation and 27 glutarylation sites—a 'dark epigenome' largely undetected by propionylation-based methods. We demonstrate that complementary digestion with Arg-C Ultra and r-Chymotrypsin provides orthogonal sequence coverage, enabling detection of PTMs in H2A variants, linker histones, and regions poorly represented by arginine-specific cleavage alone. Application of RIPUP to frozen-thawed rat hippocampal sections within a 3-hour workflow identifies >200 PTMs including biologically critical PTM sites H3 K27/K36/K37 methylation, H4 N-terminal acetylation patterns, and H2A ubiquitination at K118/K119. This rapid, high-efficiency platform enables timely discovery of epigenetic mechanisms and accelerates the path from PTM identification to therapeutic target validation.
HostingRepository	PRIDE
AnnounceDate	2026-06-20
AnnouncementXML	Submission_2026-06-19_17:04:22.963.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Natalie Turner
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606; scientific name: Rattus norvegicus (Rat); NCBI TaxID: NEWT:10116;
ModificationList	TMT6plex-126 reporter+balance reagent acylated residue
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2026-01-27 18:45:57	ID requested
⏵ 1	2026-06-19 17:04:23	announced

10.1021/acs.analchem.6c01147;

Turner NP, Baboo S, Garrett P, Diedrich JK, Bajo M, Roberto M, Yates Iii JR, Rapid Histone Post-Translational Modification Analysis Using Alternative Proteases and Tandem Mass Tags. Anal Chem, 98(25):18694-18712(2026) [pubmed]

submitter keyword: post-translational modifications,Histones, proteases, LC-MS/MS

John R Yates III
contact affiliation	Department of Integrative and Structural Computational Biology, The Scripps Research Institute, La Jolla, CA
contact email	jyates@scripps.edu
lab head
Natalie Turner
contact affiliation	The Scripps Research Institute
contact email	naturner@scripps.edu
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/2026/06/PXD073683

PRIDE project URI

• PRIDE
  1. PXD073683
     1. Label: PRIDE project
     2. Name: Rapid Histone Post-Translational Modification Analysis Using Alternative Proteases and Tandem Mass Tags (RIPUP)