# Identification of Small Open Reading Frame-Encoded Peptides in Glioma by an Optimized Proteomics Strategy

## TL;DR

Researchers developed a new method to identify short peptides in glioma, discovering 549 novel peptides, some linked to tumor progression.

## Contribution

An optimized proteomics strategy, AmF-C8-SPE, significantly improves the identification of small peptides in glioma.

## Key findings

- AmF-C8-SPE outperforms classic C8-SPE in identifying SEPs with higher unique peptide ratios and coverage.
- 549 novel SEPs were identified in glioma, 113 of which showed differential expression in tumors.
- Two SEPs (IP_613981 and SPROHSA206836) were confirmed to localize in the nucleus.

## Abstract

Small open reading frame–encoded peptides (SEPs), translated from previously unannotated genomic regions, have emerged as important regulators in diverse physiological and pathological processes. While ribosome profiling and bioinformatics analysis can predict putative SEPs, mass spectrometry (MS) is the only method for their definitive identification. However, MS-based SEP detection faces significant challenges due to SEP’s short length and low abundance. To address these limitations, we developed an ammonium formate-mediated C8 solid-phase enrichment (AmF-C8-SPE) strategy that significantly outperforms classic C8-SPE, yielding superior SEP identification with enhanced unique peptide ratios and sequence coverage. By coupling AmF-C8-SPE with fractionation and LC-MS/MS analysis of glioma samples from 18 patients, we identified 549 novel SEPs, 113 of which exhibited differential expression between tumors and adjacent normal tissues. Importantly, randomly selected SEPs were validated by MS spectral matching with synthetic peptides and by confirming recombinant fusion protein expression in cells. Furthermore, Mfuzz clustering and ROC curve analyses revealed SEPs associated with glioma progression. DeepLoc-based prediction followed by confocal microscopy imaging confirmed nuclear localization of two candidate SEPs (IP_613981 and SPROHSA206836). Therefore, this study establishes an optimized SEP identification approach and the first comprehensive SEP profiling in glioma, providing a valuable resource to discover novel glioma biomarker and therapeutic target.

•An optimized method termed AmF-C8-SPE was developed for SEP enrichment.•Coupling AmF-C8-SPE with fractionation and LC-MS/MS identified 549 SEPs in glioma.•Mfuzz clustering and ROC analysis revealed SEPs associated with glioma progression.•Two SEPs (IP_613981 and SPROHSA206836) were confirmed to localize in the nucleus.

An optimized method termed AmF-C8-SPE was developed for SEP enrichment.

Coupling AmF-C8-SPE with fractionation and LC-MS/MS identified 549 SEPs in glioma.

Mfuzz clustering and ROC analysis revealed SEPs associated with glioma progression.

Two SEPs (IP_613981 and SPROHSA206836) were confirmed to localize in the nucleus.

This study developed an optimized proteomics workflow integrating ammonium formate -mediated C8 solid - phase enrichment (AmF-C8-SPE) with fractionation and LC-MS/MS analysis to significantly improve small open reading frame–encoded peptide (SEP) identification. Compared to classic C8 solid-phase enrichment (Classic-C8), AmF-C8-SPE demonstrated superior SEP identification efficiency and data quality. Applying this approach to clinical glioma samples, we identified 549 novel SEPs, including 113 with differential expression between tumors and adjacent normal tissues, establishing the first comprehensive qualitative and quantitative SEP profiling in glioma.