Proteogenomic Discovery of a Small, Novel Protein in Yeast Reveals a Strategy for the Detection of Unannotated Short Open Reading Frames

Yagoub, Daniel; Tay, Aidan P.; Chen, Zhiliang; Hamey, Joshua J.; Cai, Curtis; Chia, Samantha Z.; Hart‐Smith, Gene; Wilkins, Marc R.

doi:10.1021/acs.jproteome.5b00734

Cited by 25 publications

(26 citation statements)

References 64 publications

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“…Non‐labelled S. cerevisiae cells, strain BY4741, were cultured according to the methods by Yagoub et al . Yeast SILAC cells were grown to ≈0.8 OD 600 nm in minimal media (1.7 g L −1 yeast nitrogen base without amino acids and ammonium sulfate, 5.6 g L −1 ammonium sulfate, 20 g L −1 glucose, 211 mg L −1 histidine, 260 mg L −1 leucine, 22.6 mg L −1 uracil and 76 mg L −1 methionine) supplemented with 76 mg L −1 of lysine and arginine (wild‐type BY4741), or 13 C 6 , 15 N 2 ‐L‐lysine and 13 C 6 , 15 N 4 ‐L‐arginine (∆Lys2/∆Arg4 in strain BY4741).…”

Section: Methodsmentioning

confidence: 99%

MS2‐Deisotoper: A Tool for Deisotoping High‐Resolution MS/MS Spectra in Normal and Heavy Isotope‐Labelled Samples

et al. 2019

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High‐resolution MS/MS spectra of peptides can be deisotoped to identify monoisotopic masses of peptide fragments. The use of such masses should improve protein identification rates. However, deisotoping is not universally used and its benefits have not been fully explored. Here, MS2‐Deisotoper, a tool for use prior to database search, is used to identify monoisotopic peaks in centroided MS/MS spectra. MS2‐Deisotoper works by comparing the mass and relative intensity of each peptide fragment peak to every other peak of greater mass, and by applying a set of rules concerning mass and intensity differences. After comprehensive parameter optimization, it is shown that MS2‐Deisotoper can improve the number of peptide spectrum matches (PSMs) identified by up to 8.2% and proteins by up to 2.8%. It is effective with SILAC and non‐SILAC MS/MS data. The identification of unique peptide sequences is also improved, increasing the number of human proteoforms by 3.7%. Detailed investigation of results shows that deisotoping increases Mascot ion scores, improves FDR estimation for PSMs, and leads to greater protein sequence coverage. At a peptide level, it is found that the efficacy of deisotoping is affected by peptide mass and charge. MS2‐Deisotoper can be used via a user interface or as a command‐line tool.

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Section: Methodsmentioning

confidence: 99%

MS2‐Deisotoper: A Tool for Deisotoping High‐Resolution MS/MS Spectra in Normal and Heavy Isotope‐Labelled Samples

et al. 2019

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“…Unlike conventional MS data analysis, this approach does not rely on a reference protein database and can therefore detect previously unannotated coding regions. Improvements in throughput and proteome coverage of MS-based proteomics has potentiated the use of protein evidence to improve gene annotation in many organisms such as Campylobacter concisus (11), Saccharomyces cerevisiae (12,13), Arabidopsis thaliana (14), mouse (9,15) and human (9,16). In contrast to these previous proteogenomics studies, our present study combines proteomics and RNA-seq for genome-wide annotation as part of an integrative workflow.…”

Section: Introductionmentioning

confidence: 99%

Proteogenomics produces comprehensive and highly accurate protein-coding gene annotation in a complete genome assembly ofMalassezia sympodialis

et al. 2017

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Complete and accurate genome assembly and annotation is a crucial foundation for comparative and functional genomics. Despite this, few complete eukaryotic genomes are available, and genome annotation remains a major challenge. Here, we present a complete genome assembly of the skin commensal yeast Malassezia sympodialis and demonstrate how proteogenomics can substantially improve gene annotation. Through long-read DNA sequencing, we obtained a gap-free genome assembly for M. sympodialis (ATCC 42132), comprising eight nuclear and one mitochondrial chromosome. We also sequenced and assembled four M. sympodialis clinical isolates, and showed their value for understanding Malassezia reproduction by confirming four alternative allele combinations at the two mating-type loci. Importantly, we demonstrated how proteomics data could be readily integrated with transcriptomics data in standard annotation tools. This increased the number of annotated protein-coding genes by 14% (from 3612 to 4113), compared to using transcriptomics evidence alone. Manual curation further increased the number of protein-coding genes by 9% (to 4493). All of these genes have RNA-seq evidence and 87% were confirmed by proteomics. The M. sympodialis genome assembly and annotation presented here is at a quality yet achieved only for a few eukaryotic organisms, and constitutes an important reference for future host-microbe interaction studies.

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“…Notably, all 175 peptide identifications were validated upon comparison of the MS/MS fragmentation patterns to that of synthetic peptides. A similar approach has been undertaken in recent studies describing identification of novel peptides (Kim et al 2014;Wilhelm et al 2014;Yagoub et al 2015).…”

Section: Validation Of Ms/ms Spectra For Novel Peptidesmentioning

confidence: 99%

Integrating transcriptomic and proteomic data for accurate assembly and annotation of genomes

et al. 2016

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Complementing genome sequence with deep transcriptome and proteome data could enable more accurate assembly and annotation of newly sequenced genomes. Here, we provide a proof-of-concept of an integrated approach for analysis of the genome and proteome of Anopheles stephensi, which is one of the most important vectors of the malaria parasite. To achieve broad coverage of genes, we carried out transcriptome sequencing and deep proteome profiling of multiple anatomically distinct sites. Based on transcriptomic data alone, we identified and corrected 535 events of incomplete genome assembly involving 1196 scaffolds and 868 protein-coding gene models. This proteogenomic approach enabled us to add 365 genes that were missed during genome annotation and identify 917 gene correction events through discovery of 151 novel exons, 297 protein extensions, 231 exon extensions, 192 novel protein start sites, 19 novel translational frames, 28 events of joining of exons, and 76 events of joining of adjacent genes as a single gene. Incorporation of proteomic evidence allowed us to change the designation of more than 87 predicted “noncoding RNAs” to conventional mRNAs coded by protein-coding genes. Importantly, extension of the newly corrected genome assemblies and gene models to 15 other newly assembled Anopheline genomes led to the discovery of a large number of apparent discrepancies in assembly and annotation of these genomes. Our data provide a framework for how future genome sequencing efforts should incorporate transcriptomic and proteomic analysis in combination with simultaneous manual curation to achieve near complete assembly and accurate annotation of genomes.

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Proteogenomic Discovery of a Small, Novel Protein in Yeast Reveals a Strategy for the Detection of Unannotated Short Open Reading Frames

Cited by 25 publications

References 64 publications

MS2‐Deisotoper: A Tool for Deisotoping High‐Resolution MS/MS Spectra in Normal and Heavy Isotope‐Labelled Samples

MS2‐Deisotoper: A Tool for Deisotoping High‐Resolution MS/MS Spectra in Normal and Heavy Isotope‐Labelled Samples

Proteogenomics produces comprehensive and highly accurate protein-coding gene annotation in a complete genome assembly ofMalassezia sympodialis

Integrating transcriptomic and proteomic data for accurate assembly and annotation of genomes

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