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Peptide analysis in human cell lines via high-resolution MRM (PRM) for the annotation of non-canonical open reading frames

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Ulrike Kusebauch

LAB HEAD: Robert L.

PROVIDER: PXD066599 | Pride | 2026-02-17

REPOSITORIES: pride

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			Action	DRS
	0712_S005_90min_250ng_HeLaS3_Pr2P2_6fmol_iRT_nc240_01.timeseries.data	Other
	0712_S005_90min_250ng_HeLaS3_Pr2P2_6fmol_iRT_nc240_01.wiff	Wiff
	0712_S005_90min_250ng_HeLaS3_Pr2P2_6fmol_iRT_nc240_01.wiff.scan	Wiff
	0712_S005_90min_250ng_HeLaS3_Pr2P2_6fmol_iRT_nc240_01.wiff2	Wiff
	0712_S006_90min_250ng_Hek293_Pr2P2_6fmol_iRT_nc240_01.timeseries.data	Other

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Peptide analysis in human cell lines via high-resolution MRM (PRM) for the annotation of non-canonical open reading frames

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