OpenProt 2.0 builds a path to the functional characterization of alternative proteins

Abstract

The OpenProt proteogenomic resource (https:////www.openprot.org//) provides users with a complete and freely accessible set of non-canonical or alternative open reading frames (AltORFs) within the transcriptome of various species, as well as functional annotations of the corresponding protein sequences not found in standard databases. Enhancements in this update are largely the result of user feedback and include the prediction of structure, subcellular localization, and intrinsic disorder, using cutting-edge algorithms based on machine learning techniques. The mass spectrometry pipeline now integrates a machine learning-based peptide rescoring method to improve peptide identification. We continue to help users explore this cryptic proteome by providing OpenCustomDB, a tool that enables users to build their own customized protein databases, and OpenVar, a genomic annotator including genetic variants within AltORFs and protein sequences. A new interface improves the visualization of all functional annotations, including a spectral viewer and the prediction of multicoding genes. All data on OpenProt are freely available and downloadable. Overall, OpenProt continues to establish itself as an important resource for the exploration and study of new proteins.

Non-canonical altPIDD1 protein: unveiling the true major translational output of thePIDD1gene

“…1A). However, a second ORF spanning exons 2 and 3 with an initiation codon upstream of PIDD1 initiation codon, is annotated by OpenProt (Leblanc et al, 2024). This alternative ORF is predicted to encode a 171-amino acid protein termed altPIDD1 (Fig.…”

“…To address this issue, several studies and proteogenomic resources combining ribo-seq and proteomics help annotate altORFs and altProts (Olexiouk et al, 2018; Li et al, 2021; Ouspenskaia et al, 2022; Manske et al, 2023; Sandmann et al, 2023). The proteogenomic resource OpenProt allows for the identification of more than one ORF per transcript and annotates all ORFs larger than 29 codons in the transcriptome of different species and their corresponding proteins (Leblanc et al, 2024). The reanalysis of large-scale ribo-seq and proteomics data with OpenProt allows for the retrieval of evidence of expression at the translatome (altORFs) and at the proteome (altProts) levels, respectively, and the identification of potential multicoding genes.…”

Biosurfer for systematic tracking of regulatory mechanisms leading to protein isoform diversity

“…Currently, the prediction of proteins from deep coverage long-read RNA-seq datasets rely on heuristics, such as prioritizing ORF from an alternative isoform that shares the same start AUG codon with the reference, or selection of the most 5' proximal AUG (Tang et al 2020;Miller et al 2022), whereas others have developed computationally efficient scoring strategy that ranks more highly the ORFs with highest protein similarity to the reference (Varabyou et al 2022(Varabyou et al , 2023. To provide more reliable ORF annotations, experimental approaches like Ribo-Seq demarcate novel coding regions, including sites of non-canonical translation, which might be information that could be incorporated in proteogenomic workflows (Mudge et al 2022;Leblanc et al 2024).…”

Biophysical characterization of high-confidence, small human proteins

“…prevalence of transmembrane helices(21, 36, 41, 42) or disorder(23). Structural predictions are available for a wide range of putative and alternative smORFs in the Openprot database(43). In contrast to other studies which have sought to catalogue large permissive sets of candidate SEPs(44, 45), we sought to characterize only high-confidence SEPs.…”