Detection of SEPs by MS (Second Round)
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Velos
ORGANISM(S): Staphylococcus Aureus
TISSUE(S): Cell Culture
SUBMITTER: 
Samuel Miravet Verde
LAB HEAD: Luis Serrano
PROVIDER: PXD011038 | Pride | 2019-10-14
REPOSITORIES: Pride
Similar Datasets
Project description:Identifying smORFs and SEPs is technically and computationally challenging. Experimentally, techniques as ribosome profiling (Ribo-Seq and mass spectroscopy (MS) are used. Ribo-Seq sequences the mRNA and does not provide the translated frame, thus identifying proteins encoded by overlapping ORFs is not feasible. Herein we have used MS to characterize smORFomes of different Mycoplasma species and Escherichia coli. This data is used to corroborate the predictions of a random forest classifier that in silico predicts all the putative SEPs encoded by different bacterial genomes.
2019-10-14 | PXD010490 | Pride
Project description:Identifying smORFs and SEPs is technically and computationally challenging. Experimentally, techniques as ribosome profiling (Ribo-Seq and mass spectroscopy (MS) are used. Ribo-Seq sequences the mRNA and does not provide the translated frame, thus identifying proteins encoded by overlapping ORFs is not feasible. Herein we have used MS to characterize smORFomes of different Mycoplasma species. This data is used to corroborate the predictions of a random forest classifier that in silico predicts all the putative SEPs encoded by different bacterial genomes.
2019-03-01 | PXD008243 | Pride
Project description:Many small open reading frames (smORFs) embedded in lncRNA transcripts have been shown to encode biologically functional polypeptides (smORFs-encoded polypeptides, SEPs) in different organisms. Despite significant advances in genomics, bioinformatics and proteomics that largely enabled the discovery of novel SEPs, their identification across different biological samples is still hampered by their poor predictability, diminutive size and low relative abundance. Here, we take advantage of NONCODE, a repository containing the most complete collection and annotation of lncRNA transcripts from different species, to build a novel database that attempts to maximize a collection of SEPs from human and mouse lncRNA transcripts. In order to further improve SEP discovery, we implemented two effective and complementary polypeptide enrichment strategies, 30 kDa MWCO filter and C8 SPE column. These combined strategies enabled us to discover 357 and 409 SEPs from, respectively, 8 human cell lines, and 3 mouse cell lines and 8 mouse tissues. Importantly, nineteen of the identified SEPs were then verified through in-vitro expression, immunoblotting, parallel reaction monitoring (PRM) and synthetic peptides. Subsequent bioinformatic analysis revealed that some of the physical and chemical properties of these novel SEPs, including amino acid composition and codon usage, are different from those commonly found in canonical proteins. Intriguingly, nearly 65% of the identified SEPs were found to be initiated with non-AUG start codons. Overall, the strategy presented in this study encompasses an efficient workflow that enabled us to identify 766 novel SEPs across multiple cell lines and tissues, which probably represents the largest number of SEPs detected by mass spectrometry reported to date. These novel SEPs might not only provide new clues for the annotation of noncoding elements in the genome but can also serve as a valuable resource for the functional characterization of individual SEPs.
2021-06-15 | PXD019486 | Pride
Project description:Protein-coding small open reading frames (smORFs) are emerging as an important class of genes, however, the coding capacity of smORFs in the human genome is unclear. By integrating de novo transcriptome assembly and Ribo-Seq, we confidently annotate thousands of novel translated smORFs in three human cell lines. We find that smORF translation prediction is noisier than for annotated coding sequences, underscoring the importance of analyzing multiple experiments and footprinting conditions. These smORFs are located within non-coding and antisense transcripts, the UTRs of mRNAs, and unannotated transcripts. Analysis of RNA levels and translation efficiency during cellular stress identifies regulated smORFs and provides an approach for identifying smORFs for further investigation. Sequence conservation and signatures of positive selection indicate that encoded microproteins are likely functional. Additionally, proteomics data from enriched human leukocyte antigen complexes validates the translation of hundreds of smORFs and positions them as a source of novel antigens. Thus, smORFs represent a significant number of important, yet unexplored human genes.
2019-07-03 | GSE125218 | GEO
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
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.
2017-10-17 | PXD005643 | Pride
Project description:Small open reading frames (smORFs) can have important regulatory roles and give rise to stable proteins, yet their discovery based on sequence-based predictions and proteomics has been challenging. Ribosome profiling (Ribo-seq) data can provide valuable experimental evidence of RNA translation. Stringent analysis of P-sites, representing 1.3 billion high-confidence ribosome locations, revealed 5308 uORFs, 1652 smORFs in lincRNAs and 807 dORFs that are translated in humans. We here provide a comprehensive database of Ribo-seq smORFs for a more complete understanding of the translated human genome.
2022-07-18 | GSE182372 | GEO
Project description:Small open reading frames (smORFs) can have important regulatory roles and give rise to stable proteins, yet their discovery based on sequence-based predictions and proteomics has been challenging. Ribosome profiling (Ribo-seq) data can provide valuable experimental evidence of RNA translation. Stringent analysis of P-sites, representing 1.3 billion high-confidence ribosome locations, revealed 5308 uORFs, 1652 smORFs in lincRNAs and 807 dORFs that are translated in humans. We here provide a comprehensive database of Ribo-seq smORFs for a more complete understanding of the translated human genome.
2022-07-18 | GSE182371 | GEO
Project description:Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.
2021-01-11 | GSE164239 | GEO
Project description:The absence of thousands of recently annotated small open reading frame (smORF)-encoded peptides and small proteins (microproteins) from databases has precluded their analysis in metabolism and metabolic disease. Given the outsized importance of small proteins and peptides in metabolism—insulin, leptin, amylin, glucagon, and glucagon-like peptide-1 (GLP-1)—microproteins are a potentially rich source of uncharacterized metabolic regulators. Here, we annotate smORFs in primary differentiated brown, white, and beige mouse adipose cells. Ribosome profiling (Ribo-Seq) detected a total of 3,877 unannotated smORFs. Analysis of RNA-Seq datasets revealed diet-regulated smORF expression in adipose tissues, and validated the adipose translation of the feeding-neuron marker gene Gm8773. Gm8773 encodes the mouse homolog of FAM237B, a neurosecretory protein that stimulates food intake and promotes weight gain in chickens. Testing of recombinant mFAM237B produced similar orexigenic activity in mice further supporting a role for FAM237B a metabolic regulator and part of the brain-adipose axis. Furthermore, we showed that data independent acquisition mass spectrometry (DIA-MS) proteomics can provide a sensitive, flexible, and quantitative platform for identifying microproteins by mass spectrometry. Using this system led to the detection of 58 microproteins from cell culture and an additional 25 from mouse serum. The proteomics data established the anti-inflammatory microprotein AW112010 as a circulating factor, and found that serum levels of a microprotein translated from a FRS2 uORF is elevated in older obese mice. Together, the data highlight the value of this database in examining understudied smORFs and microproteins in metabolic research and identifying additional regulators of metabolism.
2022-12-13 | GSE198107 | GEO