Browse
Submit Data
Databases
API
Help

Dataset Information

20 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Improved Identification of Small Open Reading Frames Encoded Peptides by Top-Down Proteomic Approaches and De Novo Sequencing.

ABSTRACT: Small open reading frames (sORFs) have translational potential to produce peptides that play essential roles in various biological processes. Nevertheless, many sORF-encoded peptides (SEPs) are still on the prediction level. Here, we construct a strategy to analyze SEPs by combining top-down and de novo sequencing to improve SEP identification and sequence coverage. With de novo sequencing, we identified 1682 peptides mapping to 2544 human sORFs, which were all first characterized in this work. Two-thirds of these new sORFs have reading frame shifts and use a non-ATG start codon. The top-down approach identified 241 human SEPs, with high sequence coverage. The average length of the peptides from the bottom-up database search was 19 amino acids (AA); from de novo sequencing, it was 9 AA; and from the top-down approach, it was 25 AA. The longer peptide positively boosts the sequence coverage, more efficiently distinguishing SEPs from the known gene coding sequence. Top-down has the advantage of identifying peptides with sequential K/R or high K/R content, which is unfavorable in the bottom-up approach. Our method can explore new coding sORFs and obtain highly accurate sequences of their SEPs, which can also benefit future function research.

SUBMITTER: Wang B

PROVIDER: S-EPMC8197016 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Top-down analysis of protein samples by de novo sequencing techniques.

Project description:MOTIVATION:Recent technological advances have made high-resolution mass spectrometers affordable to many laboratories, thus boosting rapid development of top-down mass spectrometry, and implying a need in efficient methods for analyzing this kind of data. RESULTS:We describe a method for analysis of protein samples from top-down tandem mass spectrometry data, which capitalizes on de novo sequencing of fragments of the proteins present in the sample. Our algorithm takes as input a set of de novo amino acid strings derived from the given mass spectra using the recently proposed Twister approach, and combines them into aggregated strings endowed with offsets. The former typically constitute accurate sequence fragments of sufficiently well-represented proteins from the sample being analyzed, while the latter indicate their location in the protein sequence, and also bear information on post-translational modifications and fragmentation patterns. AVAILABILITY AND IMPLEMENTATION:Freely available on the web at http://bioinf.spbau.ru/en/twister CONTACT:vyatkina@spbau.ru or ppevzner@ucsd.edu SUPPLEMENTARY INFORMATION:Supplementary data are available at Bioinformatics online.

| S-EPMC6280873 | biostudies-other

Characterization of Chlamydia trachomatis plasmid-encoded open reading frames.

Project description:The recent success in transformation of Chlamydia trachomatis represents a major advancement in Chlamydia research. Plasmid-free C. trachomatis serovar L2 organisms can be transformed with chlamydial plasmid-based shuttle vectors pGFP::SW2 and pBRCT. Deletion of plasmid genes coding for Pgp1 to Pgp8 in pBRCT led to the identification of Pgp1, -2, -6, and -8 as plasmid maintenance factors; Pgp4 as a transcriptional regulator of chlamydial virulence-associated gene expression; and Pgp3, -5, and -7 as being dispensable for chlamydial growth in vitro. Using the pGFP::SW2 vector system, we confirmed these findings in the current report. To further dissect the roles of pgp coding sequences and Pgp proteins in plasmid maintenance, we introduced premature stop codons into the pgp genes. Stable transformants were obtained with pGFP::SW2 derivatives carrying premature stop codons in pgp8 but not in pgp1, pgp2, and pgp6, suggesting that the pgp8 coding sequence but not the Pgp8 protein is required for maintaining the plasmid, while Pgp1, -2, and -6 proteins are necessary for plasmid maintenance. We also found that a minimum of 30 nucleotides in the pgp3 coding region was required for pgp4 expression. Finally, mCherry red fluorescent protein was successfully expressed when the mCherry gene was used to replace the pgp3, pgp4, or pgp5 coding region, indicating that these regions can be used to express nonchlamydial genes in chlamydial organisms. These novel observations have provided information for further use of chlamydial plasmid shuttle vectors as genetic tools to understand chlamydial biology and pathogenicity as well as to develop attenuated chlamydial vaccines.

| S-EPMC3754608 | biostudies-literature

Analysis of nested alternate open reading frames and their encoded proteins.

Project description:Transcriptional and post-transcriptional mechanisms diversify the proteome beyond gene number, while maintaining a sequence relationship between original and altered proteins. A new mechanism breaks this paradigm, generating novel proteins by translating alternative open reading frames (Alt-ORFs) within canonical host mRNAs. Uniquely, 'alt-proteins' lack sequence homology with host ORF-derived proteins. We show global amino acid frequencies, and consequent biochemical characteristics of Alt-ORFs nested within host ORFs (nAlt-ORFs), are genetically-driven, and predicted by summation of frequencies of hundreds of encompassing host codon-pairs. Analysis of 101 human nAlt-ORFs of length ≥150 codons confirms the theoretical predictions, revealing an extraordinarily high median isoelectric point (pI) of 11.68, due to anomalous charged amino acid levels. Also, nAlt-ORF proteins exhibit a >2-fold preference for reading frame 2 versus 3, predicted mitochondrial and nuclear localization, and elevated codon adaptation index indicative of natural selection. Our results provide a theoretical and conceptual framework for exploration of these largely unannotated, but potentially significant, alternative ORFs and their encoded proteins.

| S-EPMC9580016 | biostudies-literature

Top-Down Proteomic Characterization of Truncated Proteoforms.

Project description:A top-down proteomic strategy with semiautomated analysis of data sets has proven successful for the global identification of truncated proteins without the use of chemical derivatization, enzymatic manipulation, immunoprecipitation, or other enrichment. This approach provides the reliable identification of internal polypeptides formed from precursor gene products by proteolytic cleavage of both the N- and C-termini, as well as truncated proteoforms that retain one or the other termini. The strategy has been evaluated by application to the immunosuppressive extracellular vesicles released by myeloid-derived suppressor cells. More than 1000 truncated proteoforms have been identified, from which binding motifs are derived to allow characterization of the putative proteases responsible for truncation.

| S-EPMC7029676 | biostudies-literature

Neutron-encoded mass signatures for quantitative top-down proteomics.

Project description:The ability to acquire highly accurate quantitative data is an increasingly important part of any proteomics experiment, whether shotgun or top-down approaches are used. We recently developed a quantitation strategy for peptides based on neutron encoding, or NeuCode SILAC, which uses closely spaced heavy isotope-labeled amino acids and high-resolution mass spectrometry to provide quantitative data. We reasoned that the strategy would also be applicable to intact proteins and could enable robust, multiplexed quantitation for top-down experiments. We used yeast lysate labeled with either (13)C6(15)N2-lysine or (2)H8-lysine, isotopologues of lysine that are spaced 36 mDa apart. Proteins having such close spacing cannot be distinguished during a medium resolution scan, but upon acquiring a high-resolution scan, the two forms of the protein with each amino acid are resolved and the quantitative information revealed. An additional benefit NeuCode SILAC provides for top down is that the spacing of the isotope peaks indicates the number of lysines present in the protein, information that aids in identification. We used NeuCode SILAC to quantify several hundred isotope distributions, manually identify and quantify proteins from 1:1, 3:1, and 5:1 mixed ratios, and demonstrate MS(2)-based quantitation using ETD.

| S-EPMC3983007 | biostudies-literature

"De-novo" amino acid sequence elucidation of protein G'e by combined "top-down" and "bottom-up" mass spectrometry.

Project description:Mass spectrometric de-novo sequencing was applied to review the amino acid sequence of a commercially available recombinant protein G´ with great scientific and economic importance. Substantial deviations to the published amino acid sequence (Uniprot Q54181) were found by the presence of 46 additional amino acids at the N-terminus, including a so-called "His-tag" as well as an N-terminal partial α-N-gluconoylation and α-N-phosphogluconoylation, respectively. The unexpected amino acid sequence of the commercial protein G' comprised 241 amino acids and resulted in a molecular mass of 25,998.9 ± 0.2 Da for the unmodified protein. Due to the higher mass that is caused by its extended amino acid sequence compared with the original protein G' (185 amino acids), we named this protein "protein G'e." By means of mass spectrometric peptide mapping, the suggested amino acid sequence, as well as the N-terminal partial α-N-gluconoylations, was confirmed with 100% sequence coverage. After the protein G'e sequence was determined, we were able to determine the expression vector pET-28b from Novagen with the Xho I restriction enzyme cleavage site as the best option that was used for cloning and expressing the recombinant protein G'e in E. coli. A dissociation constant (K(d)) value of 9.4 nM for protein G'e was determined thermophoretically, showing that the N-terminal flanking sequence extension did not cause significant changes in the binding affinity to immunoglobulins.

| S-EPMC6130978 | biostudies-other

De Novo Sequencing of Top-Down Tandem Mass Spectra: A Next Step towards Retrieving a Complete Protein Sequence.

Project description:De novo sequencing of tandem (MS/MS) mass spectra represents the only way to determine the sequence of proteins from organisms with unknown genomes, or the ones not directly inscribed in a genome-such as antibodies, or novel splice variants. Top-down mass spectrometry provides new opportunities for analyzing such proteins; however, retrieving a complete protein sequence from top-down MS/MS spectra still remains a distant goal. In this paper, we review the state-of-the-art on this subject, and enhance our previously developed Twister algorithm for de novo sequencing of peptides from top-down MS/MS spectra to derive longer sequence fragments of a target protein.

| S-EPMC5372227 | biostudies-literature

Peptides derived from small mitochondrial open reading frames: Genomic, biological, and therapeutic implications.

Project description:Mitochondrial-derived peptides (MDPs) are a novel class of bioactive microproteins that modify cell metabolism. The the eight MDPs that been characterized (e.g., humanin, MOTS-c, SHLPs1-6) attenuate disease pathology including Alzheimer's disease, prostate cancer, macular degeneration, cardiovascular disease, and diabetes. The association between disease and human genetic variation in MDPs is underexplored, although two polymorphisms in humanin and MOTS-c associate with cognitive decline and diabetes, respectively, suggesting a precise role for MDPs in disease-modification. There could be hundreds of additional MDPs that have yet to be discovered. Altogether, MDPs could explain unanswered biological and metabolic questions and are part of a growing field of novel microproteins encoded by small open reading frames. In this review, the current state of MDPs are summarized with an emphasis on biological and therapeutic implications.

| S-EPMC7778388 | biostudies-literature

Hydrophobic Alpha-Helical Short Peptides in Overlapping Reading Frames of the Coronavirus Genome.

Project description:In this study, we show that the coronavirus (CoV) genome may encode many functional hydrophobic alpha-helical peptides (HAHPs) in overlapping reading frames of major coronaviral proteins throughout the entire viral genome. These HAHPs can theoretically be expressed from non-canonical sub-genomic (sg)RNAs that are synthesized in substantial amounts in infected cells. We selected and analyzed five and six HAHPs encoded in the S gene regions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively. Two and three HAHPs derived from SARS-CoV-2 and MERS-CoV, respectively, specifically interacted with both the SARS-CoV-2 and MERS-CoV S proteins and inhibited their membrane fusion activity. Furthermore, one of the SARS-CoV-2 HAHPs specifically inhibited viral RNA synthesis by accumulating at the site of viral RNA synthesis. Our data show that a group of HAHPs in the coronaviral genome potentially has a regulatory role in viral propagation.

| S-EPMC9415614 | biostudies-literature

Reading therapy strengthens top-down connectivity in patients with pure alexia.

Project description:This study tested the efficacy of audio-visual reading training in nine patients with pure alexia, an acquired reading disorder caused by damage to the left ventral occipitotemporal cortex. As well as testing the therapy's impact on reading speed, we investigated the functional reorganization underlying therapy-induced behavioural changes using magnetoencephalography. Reading ability was tested twice before training (t1 and t2) and twice after completion of the 6-week training period (t3 and t4). At t3 there was a significant improvement in word reading speed and reduction of the word length effect for trained words only. Magnetoencephalography at t3 demonstrated significant differences in reading network connectivity for trained and untrained words. The training effects were supported by increased bidirectional connectivity between the left occipital and ventral occipitotemporal perilesional cortex, and increased feedback connectivity from the left inferior frontal gyrus. Conversely, connection strengths between right hemisphere regions became weaker after training.

| S-EPMC3722354 | biostudies-literature

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