Browse
Submit Data
Databases
API
Help

Dataset Information

22 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Small Protein Enrichment Improves Proteomics Detection of sORF Encoded Polypeptides

ABSTRACT: With the rapid growth in the number of sequenced genomes, genome annotation efforts became almost exclusively reliant on automated pipelines. Despite their unquestionable utility, these methods have been shown to underestimate the true complexity of the studied genomes, with small open reading frames (sORFs; ORFs typically considered shorter than 300 nucleotides) and, in consequence, their protein products (sORF encoded polypeptides or SEPs) being the primary example of a poorly annotated and highly underexplored class of genomic elements. With the advent of advanced translatomics such as ribosome profiling, reannotation efforts have progressed a great deal in providing translation evidence for numerous, previously unannotated sORFs. However, proteomics validation of these riboproteogenomics discoveries remains challenging due to their short length and often highly variable physiochemical properties. In this work we evaluate and compare tailored, yet easily adaptable, protein extraction methodologies for their efficacy in the extraction and concomitantly proteomics detection of SEPs expressed in the prokaryotic model pathogen Salmonella typhimurium (S. typhimurium). Further, an optimized protocol for the enrichment and efficient detection of SEPs making use of the of amphipathic polymer amphipol A8-35 and relying on differential peptide vs. protein solubility was developed and compared with global extraction methods making use of chaotropic agents. Given the versatile biological functions SEPs have been shown to exert, this work provides an accessible protocol for proteomics exploration of this fascinating class of small proteins.

SUBMITTER: Fijalkowski I

PROVIDER: S-EPMC8554064 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Small protein enrichment improves proteomics detection of sORF encoded peptides.

Project description:With the rapid growth in the number of sequenced genomes, genome annotation efforts became almost exclusively reliant on automated pipelines. Despite their unquestionable utility, these methods have been shown to underestimate the true complexity of the studied genomes, with small open reading frames (sORFs; ORFs shorter than 300 nucleotides) and, in consequence, their protein products (sORF encoded polypeptides or SEPs) being the primary example of a poorly annotated and highly underexplored class of genomic elements. With the advent of advanced translatomics such as ribosome profiling, reannotation efforts have progressed a great deal in providing translation evidence for numerous, previously unannotated sORFs. However, proteomics validation of these riboproteogenomics discoveries remains challenging due to their short length and often highly variable physiochemical properties. In this work we evaluate and compare tailored, yet easily adaptable, protein extraction methodologies for their efficacy in facilitating the extraction and concomitantly proteomics detection of SEPs expressed in the prokaryotic model organism Salmonella Typhimurium. An optimized protocol for the enrichment and efficient detection of SEPs making use of the of amphipathic polymer amphipol A8-35 relying on the differential peptide versus protein solubility is suggested and compared with global extraction methods making use of chaotropic agents. Given the versatile biological functions the SEPs have been shown to exert, this work provides an accessible protocol for proteomics exploration of this fascinating class of small proteins.

2021-10-07 | PXD025297 | Pride

Discovery of human sORF-encoded polypeptides (SEPs) in cell lines and tissue.

Project description:The existence of nonannotated protein-coding human short open reading frames (sORFs) has been revealed through the direct detection of their sORF-encoded polypeptide (SEP) products. The discovery of novel SEPs increases the size of the genome and the proteome and provides insights into the molecular biology of mammalian cells, such as the prevalent usage of non-AUG start codons. Through modifications of the existing SEP-discovery workflow, we discover an additional 195 SEPs in K562 cells and extend this methodology to identify novel human SEPs in additional cell lines and human tissue for a final tally of 237 new SEPs. These results continue to expand the human genome and proteome and demonstrate that SEPs are a ubiquitous class of nonannotated polypeptides that require further investigation.

| S-EPMC3993966 | biostudies-literature

Hidden in plain sight; challenges in proteomics detection of small ORF-encoded polypeptides.

Project description:Genomic studies of bacteria have long pointed towards widespread prevalence of small open reading frames (sORFs) encoding for short proteins, less than 100 amino acids in length. Despite the mounting genomic evidence of their robust expression, little progress has been made in their mass spectrometry-based validation. Various blanket statements have been used to explain this discrepancy with the potentially low expression levels and protein instability being put forward most often. In this study we provide the first systemic investigation of the challenging nature of proteomic detection of such small proteins. A panel of physiochemical properties alongside recently developed mass spectrometry detectability metrics is interrogated to provide the first evidence-based study of SEP detectability. Moreover, a large scale proteomics and genomics compendium of proteins produced by Salmonella Typhimurium (S. Typhimurium), a model human pathogen, across a panel of growth conditions is presented and used to further support our in silico analysis. This integrative riboproteogenomic approach is used to provide a data-driven census of small proteins expressed by S. Typhimurium across growth phases and infection-relevant conditions. Taken together our study delineates current limitations in proteomics based validation of novel small proteins currently missing from bacterial genome annotations.

2023-07-20 | PXD029391 | Pride

Comprehensive peptide analysis of mouse brain striatum identifies novel sORF-encoded polypeptides

Project description:Bio-active peptides are involved in the regulation of most if not all physiological processes in the body. Classical bio-active peptides (CBAPs) are mostly cleaved from a larger precursor protein and stored in secretion vesicles from which they are released in the extracellular space to exert there (signaling) activity. Only recently, another non-classical type of bio-active peptides (NCBAPs) is being discovered. These typically are not secreted but instead appear to be translated from short open reading frames (sORF) and released directly into the cytoplasm. In contrast to CBAPs, they appear to be involved in the regulation of intra-cellular processes such as transcriptional control, calcium handling and DNA repair. However, bio-chemical evidence for the translation of these sORFs remains elusive. Comprehensive analysis of these sORF-encoded polypeptides (SEPs) by peptidomic approach is hampered by a number of methodological and biological challenges: the low molecular mass of SEPs (many are in the range of 4-10 KDa), their low abundance, their transient expression and many complications in data analysis to confidently identify these peptides. In this study, we developed a strategy to address these issues as much as possible. Since translation of these predicted sORFs is still controversial, our data-analysis strategy is aimed not to maximize the number of identifications but rather to exclude as much as possible false positive identifications. Applying this strategy to a mouse brain striatum sample, we identified 981 neuropeptides originated from 50 known (neuro)peptide precursors, demonstrating the sensitivity of the method applied. In addition, five SEPs were identified including NoBody, a SEP that was previously discovered in humans and four novel SEPS from 5’ untranslated transcript regions (UTRs).

2018-05-10 | PXD008526 | Pride

Improved Identification and Analysis of Small Open Reading Frame Encoded Polypeptides.

Project description:Computational, genomic, and proteomic approaches have been used to discover nonannotated protein-coding small open reading frames (smORFs). Some novel smORFs have crucial biological roles in cells and organisms, which motivates the search for additional smORFs. Proteomic smORF discovery methods are advantageous because they detect smORF-encoded polypeptides (SEPs) to validate smORF translation and SEP stability. Because SEPs are shorter and less abundant than average proteins, SEP detection using proteomics faces unique challenges. Here, we optimize several steps in the SEP discovery workflow to improve SEP isolation and identification. These changes have led to the detection of several new human SEPs (novel human genes), improved confidence in the SEP assignments, and enabled quantification of SEPs under different cellular conditions. These improvements will allow faster detection and characterization of new SEPs and smORFs.

| S-EPMC4939623 | biostudies-literature

Genetically Encoded Cholesterol-Modified Polypeptides.

Project description:Biological systems use post-translational modifications (PTMs) to control the structure, location, and function of proteins after expression. Despite the ubiquity of PTMs in biology, their use to create genetically encoded recombinant biomaterials is limited. We have utilized a natural lipidation PTM (hedgehog-mediated cholesterol modification of proteins) to create a class of hybrid biomaterials called cholesterol-modified polypeptides (CHaMPs) that exhibit programmable self-assembly at the nanoscale. To demonstrate the biomedical utility of CHaMPs, we used this approach to append cholesterol to biologically active peptide exendin-4 that is an approved drug for the treatment of type II diabetes. The exendin-cholesterol conjugate self-assembled into micelles, and these micelles activate the glucagon-like peptide-1 receptor with a potency comparable to that of current gold standard treatments.

| S-EPMC6693588 | biostudies-literature

Large-scale docking predicts that sORF-encoded peptides may function through protein-peptide interactions in Arabidopsis thaliana.

Project description:Several recent studies indicate that small Open Reading Frames (sORFs) embedded within multiple eukaryotic non-coding RNAs can be translated into bioactive peptides of up to 100 amino acids in size. However, the functional roles of the 607 Stress Induced Peptides (SIPs) previously identified from 189 Transcriptionally Active Regions (TARs) in Arabidopsis thaliana remain unclear. To provide a starting point for functional annotation of these plant-derived peptides, we performed a large-scale prediction of peptide binding sites on protein surfaces using coarse-grained peptide docking. The docked models were subjected to further atomistic refinement and binding energy calculations. A total of 530 peptide-protein pairs were successfully docked. In cases where a peptide encoded by a TAR is predicted to bind at a known ligand or cofactor-binding site within the protein, it can be assumed that the peptide modulates the ligand or cofactor-binding. Moreover, we predict that several peptides bind at protein-protein interfaces, which could therefore regulate the formation of the respective complexes. Protein-peptide binding analysis further revealed that peptides employ both their backbone and side chain atoms when binding to the protein, forming predominantly hydrophobic interactions and hydrogen bonds. In this study, we have generated novel predictions on the potential protein-peptide interactions in A. thaliana, which will help in further experimental validation.

| S-EPMC6188750 | biostudies-literature

ARA-PEPs: a repository of putative sORF-encoded peptides in Arabidopsis thaliana.

Project description:Many eukaryotic RNAs have been considered non-coding as they only contain short open reading frames (sORFs). However, there is increasing evidence for the translation of these sORFs into bioactive peptides with potent signaling, antimicrobial, developmental, antioxidant roles etc. Yet only a few peptides encoded by sORFs are annotated in the model organism Arabidopsis thaliana.To aid the functional annotation of these peptides, we have developed ARA-PEPs (available at http://www.biw.kuleuven.be/CSB/ARA-PEPs ), a repository of putative peptides encoded by sORFs in the A. thaliana genome starting from in-house Tiling arrays, RNA-seq data and other publicly available datasets. ARA-PEPs currently lists 13,748 sORF-encoded peptides with transcriptional evidence. In addition to existing data, we have identified 100 novel transcriptionally active regions (TARs) that might encode 341 novel stress-induced peptides (SIPs). To aid in identification of bioactivity, we add functional annotation and sequence conservation to predicted peptides.To our knowledge, this is the largest repository of plant peptides encoded by sORFs with transcript evidence, publicly available and this resource will help scientists to effortlessly navigate the list of experimentally studied peptides, the experimental and computational evidence supporting the activity of these peptides and gain new perspectives for peptide discovery.

| S-EPMC5240266 | biostudies-literature

The sORF-Encoded Peptides, ATP Synthase Subunits, Facilitate WSSV Duplication in Shrimp.

Project description:Short open reading frames (sORFs) are a newly identified family of genes, and the functions of most sORF genes and their encoded peptides (SEPs) are still unknown. In this study, two ATP synthase subunits were identified in kuruma shrimp (Marsupenaeus japonicus) as SEPs, namely MjATP5I and MjATP5L. They were widely distributed in all of the tested tissues of shrimp and upregulated in hemocytes and intestines in response to WSSV challenge. The injection of recombinant proteins (rMjATP5I and rMjATP5L) increased the expression of Ie1 and Vp28, while the knockdown of MjATP5I and MjATP5L decreased the expression of Ie1 and Vp28. All of the results suggest that MjATP5I and MjATP5L were beneficial for WSSV replication. Further exploration found that MjATP5I and MjATP5L RNAi significantly improved the shrimp survival rates, reduced ATP production, and upregulated the expression of antimicrobial peptide genes post viral challenge, and the two ATPase subunits and Relish negatively regulated each other. These results reveal that MjATP5I and MjATP5L facilitated WSSV duplication by regulating the production of ATP contents and the expression of antimicrobial peptide genes in shrimp.

| S-EPMC9692589 | biostudies-literature

Improved Identification and Analysis of Small Open Reading Frame Encoded Polypeptides

Project description:Computational, genomic, and proteomic approaches have been used to discover nonannotated proteincoding small open reading frames (smORFs). Some novel smORFs have crucial biological roles in cells and organisms, which motivates the search for additional smORFs. Proteomic smORF discovery methods are advantageous because they detect smORF-encoded polypeptides (SEPs) to validate smORF translation and SEP stability. Because SEPs are shorter and less abundant than average proteins, SEP detection using proteomics faces unique challenges. Here, we optimize several steps in the SEP discovery workflow to improve SEP isolation and identification. These changes have led to the detection of several new human SEPs (novel human genes), improved confidence in the SEP assignments, and enabled quantification of SEPs under different cellular conditions. These improvements will allow faster detection and characterization of new SEPs and smORFs.

2019-06-26 | MSV000084014 | MassIVE

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data