PXD066599-2

PXD066599 is an original dataset announced via ProteomeXchange.

Title	Peptide analysis in human cell lines via high-resolution MRM (PRM) for the annotation of non-canonical open reading frames
Description	A major scientific drive is to characterize the protein-coding genome, which is a primary basis for studying human health. But the fundamental question remains: what has been missed in prior analyses? Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states, with major implications for proteomics, genomics, and clinical science. However, the impact of ncORFs has been limited by the absence of a generalized understanding of their contribution to the human proteome. Here, we report the collaborative efforts of stakeholders in proteomics, immunopeptidomics, ORF discovery, and gene annotation to produce a consensus landscape of protein-level evidence for ncORFs. We show that at least 25% of a set of 7,264 ncORFs give rise to translated gene products detected in a large-scale analysis encompassing 3.8 billion mass spectra from 95,520 experiments. With these data, we developed an annotation framework for ncORFs and codified public tools for researchers through GENCODE and PeptideAtlas. To probe the biological implications of ncORFs, we created a custom evolutionary analysis approach, termed ORF Relative Branch Length (ORBL), and found that a substantial fraction of ncORFs exhibit evolutionary constraint as open reading frames, which associates with the chance of observing ncORF-derived peptides. To classify such cases, we invoked the model of “peptideins” as a new protein annotation concept referring to ncORF-derived polypeptides that fall short of the definition of a conventional protein. We then employed knockout screens of putative peptideins and characterized one ncORF from the OLMALINC transcript as responsible for a pan-essential cellular phenotype. Overall, our work will provide a platform to advance ncORF-derived proteins in biomedical discovery and, beyond humans, diverse animals and plants where ncORFs are similarly observed. We demonstrated that ncORF peptides are detectable in human cell lysates using targeted mass spectrometry and heavy labeled synthetic peptides. Data were acquired with a ZenoTOF 8600 as well as an Orbitrap Astral mass spectrometer.
HostingRepository	PRIDE
AnnounceDate	2026-02-24
AnnouncementXML	Submission_2026-02-24_02:41:03.017.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Ulrike Kusebauch
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606;
ModificationList	isotope labeled residue; iodoacetamide derivatized residue
Instrument	Orbitrap Astral; ZenoTOF 8600

Revision	Datetime	Status	ChangeLog Entry
0	2025-07-25 18:52:11	ID requested
1	2026-02-17 13:42:45	announced
⏵ 2	2026-02-24 02:41:03	announced	2026-02-24: Updated project metadata.

Dataset with its publication pending

ProteomeXchange project tag: string

submitter keyword: cell lines, MRMHR (PRM),human, non-canonical open reading frames

Robert L.
contact affiliation	Institute for Systems Biology, Seattle, WA, USA
contact email	rmoritz@systemsbiology.org
lab head
Ulrike Kusebauch
contact affiliation	Institute for Systems Biology, Seattle, WA, USA
contact email	ukusebauch@systemsbiology.org
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/2026/02/PXD066599

PRIDE project URI

• PRIDE
  1. PXD066599
     1. Label: PRIDE project
     2. Name: Peptide analysis in human cell lines via high-resolution MRM (PRM) for the annotation of non-canonical open reading frames