Dataset Information

Identification and analysis of small proteins and short open reading frame encoded peptides in Hep3B cell

ABSTRACT: The small proteins and short open reading frames encoded peptides (SEPs) are of fundamental importance because of their essential roles in biological processes. However, the annotation or identification of them is challenging, in part owing to the limitation of the traditional genome annotation pipeline and their inherent characteristics of low abundance and low molecular weight. To discover and characterize SEPs in Hep3B cell line, we developed an optimized peptidomic assay by combining different peptide extraction and separation methods. The organic solvent precipitation method in peptidomic showed promotion in the enrichment of low molecular proteins or peptides, and the data clearly showed a beneficial effect from the reduction of sample complexity, resulting in high-quality MS/MS spectra. Furthermore, different strategies exhibited good complementarity in improving the total amount of small proteins and their sequence coverage. In total, 1192 proteins within less than 100 amino acids were identified, including 271 newly discovered SEPs that been annotated in the OpenProt database and 147 SEPs of them encoded from ncRNA or lincRNA. Results in this work provide robust evidence to date that the human proteome is more complicated than previously appreciated, and this will be a benefit to discoveries of proteins without function annotation. SIGNIFICANCE: In this work, methods were optimized to identify SEPs in Hep3B. The organic solvent precipitation presents promotion in enrichment of low molecular proteins or peptides, and the data clearly showed a beneficial effect from the reduction of sample complexity, resulting in high quality MS/MS spectra. Different strategies exhibited good complementarity in improving total amount of small proteins and their sequence coverage. In total, 1192 proteins within less than 100 amino acids were identified, including 271 newly discovered SEPs that been annotated in the OpenProt database and 147 SEPs of them encoded from ncRNA or lincRNA. Furthermore, 22 SEPs generated from the uORF may has potential effect in translation control, and 149 newly identified SEPs have known functional domains or cross-species conservation. Results in this work present robust evidence for the coding potential of the ignored region of human genomes and may provide additional insights into tumor biology.

ORGANISM(S): Homo Sapiens

SUBMITTER: Cuihong Wan

PROVIDER: PXD025813 | iProX | Wed May 05 00:00:00 BST 2021

REPOSITORIES: iProX

ACCESS DATA

Publications

Identification and analysis of small proteins and short open reading frame encoded peptides in Hep3B cell.

Wang Bing B Hao Junhui J Pan Ni N Wang Zhiwei Z Chen Yinxuan Y Wan Cuihong C

Journal of proteomics 20200903

The small proteins and short open reading frames encoded peptides (SEPs) are of fundamental importance because of their essential roles in biological processes. However, the annotation or identification of them is challenging, in part owing to the limitation of the traditional genome annotation pipeline and their inherent characteristics of low abundance and low molecular weight. To discover and characterize SEPs in Hep3B cell line, we developed an optimized peptidomic assay by combining differe ...[more]

PMID: 32891891

Similar Datasets

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.

Project description:Long non-coding RNAs (lncRNAs) are generally defined as RNA transcripts longer than 200 nucleotides that are not translated into proteins. Recently, many small open reading frames (smORFs) embedded in lncRNA scripts have been verified to be able to encode functional polypeptides (namely lncRNA-SEPs here). Although collaborative analysis by advanced genomics, bioinformatics and proteomics largely drives SEPs discovery, the poor predictability, diminutive size and low abundance still challenge systematic identification of SEPs from different biological samples. Here, we took advantage of the NONCODE database that deposited with the most complete collection and annotation of lncRNA transcripts from different species to build a database that to maximally collect all putative small ORFs from human and mouse lncRNA transcripts. Two effective and complementary polypeptides enrichment strategies (30 kDa MWCO filter and C8 SPE column) were also integrated to further improve the discovery of novel lncRNA-SEPs. These efforts led to the discovery of 362 lncRNA-SEPs from 8 human cell lines and 238 lncRNA-SEPs from 3 mouse cell lines and 8 mouse tissues. 18 out of these lncRNA-SEPs were verified experimentally by multiple technologies including in vitro expression, immunoblotting and parallel reaction monitoring-based mass spectrometry (PRM-MS) in 293T cells. Further bioinformatic analysis reveals that the physical and chemical properties of these novel lncRNA-SEPs, such as amino acid composition and codon usage, are varied from canonical proteins. Intriguingly, nearly 70% of the identified lncRNA-SEPs were found to be initiated with non-AUG start codons. Collectively, the efficient workflows presented in this study enables us identify 600 novel lncRNA-SPEs from multiple cell lines and tissues, which should represent the largest number of MS-detected lncRNA-encoding SEPs ever reported to date. These novel lncRNA-SEPs not only could provide new clues for the annotation of the noncoding elements in the genome, but also could serve as a valuable resource for the functional characterization of individual lncRNA-SEPs.

Dataset Information

Identification and analysis of small proteins and short open reading frame encoded peptides in Hep3B cell

Publications

Identification and analysis of small proteins and short open reading frame encoded peptides in Hep3B cell.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure