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Deep proteome coverage based on ribosome profiling aids mass spectrometry-based protein and peptide discovery and provides evidence of alternative translation products and near-cognate translation initiation events

(2013) MOLECULAR & CELLULAR PROTEOMICS. 12(7). p.1780-1790
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Bioinformatics: from nucleotids to networks (N2N)
Abstract
An increasing number of studies involve integrative analysis of gene and protein expression data, taking advantage of new technologies such as next-generation transcriptome sequencing (RNA-Seq) and highly sensitive mass spectrometry (MS) instrumentation. Recently, a strategy, termed ribosome profiling (or RIBO-seq), based on deep sequencing of ribosome-protected mRNA fragments, indirectly monitoring protein synthesis, has been described. We devised a proteogenomic approach constructing a custom protein sequence search space, built from both SwissProt and RIBO-seq derived translation products, applicable for MS/MS spectrum identification. To record the impact of using the constructed deep proteome database we performed two alternative MS-based proteomic strategies: (I) a regular shotgun proteomic and (II) an N-terminal COFRADIC approach. While the former technique gives an overall assessment on the protein and peptide level, the latter technique, specifically enabling the isolation of N-terminal peptides, is very appropriate in validating the RIBO-seq derived (alternative) translation initiation site profile. We demonstrate that this proteogenomic approach increases the overall protein identification rate with 2.5% (e.g. new protein products, new protein splice variants, SNP variant proteins, and N-terminally extended forms of known proteins) as compared to only searching UniProtKB-SwissProt. Furthermore, using this custom database, identification of N-terminal COFRADIC data resulted in detection of 16 alternative start sites giving rise to N-terminally extended protein variants besides the identification of four translated uORFs. Notably, the characterization of these new translation products revealed the use of multiple near-cognate (non-AUG) start codons. As deep sequencing techniques are becoming more standard, less expensive, and widespread, we anticipate that mRNA-seq and especially custom-tailored RIBO-seq will become indispensible in the MS-based protein or peptide identification process.
Keywords
upstream translation, ribosome, downstream translation, profiling, start site, near-­‐cognate, start codon, RIBO-­‐seq, N-­‐terminal, COFRADIC, Shotgun proteomics, proteogenomics, OPEN READING FRAMES, FRACTIONAL DIAGONAL CHROMATOGRAPHY, N-TERMINAL PEPTIDES, RNA-SEQ DATA, MESSENGER-RNA, SUBCELLULAR-LOCALIZATION, GENE-EXPRESSION, HUMAN-CELLS, IN-VIVO, GENERATION

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Citation

Please use this url to cite or link to this publication:

Chicago
Menschaert, Gerben, Wim Van Criekinge, Tineke Notelaers, Alexander Koch, Jeroen Crappé, Kris Gevaert, and Petra Van Damme. 2013. “Deep Proteome Coverage Based on Ribosome Profiling Aids Mass Spectrometry-based Protein and Peptide Discovery and Provides Evidence of Alternative Translation Products and Near-cognate Translation Initiation Events.” Molecular & Cellular Proteomics 12 (7): 1780–1790.
APA
Menschaert, G., Van Criekinge, W., Notelaers, T., Koch, A., Crappé, J., Gevaert, K., & Van Damme, P. (2013). Deep proteome coverage based on ribosome profiling aids mass spectrometry-based protein and peptide discovery and provides evidence of alternative translation products and near-cognate translation initiation events. MOLECULAR & CELLULAR PROTEOMICS, 12(7), 1780–1790.
Vancouver
1.
Menschaert G, Van Criekinge W, Notelaers T, Koch A, Crappé J, Gevaert K, et al. Deep proteome coverage based on ribosome profiling aids mass spectrometry-based protein and peptide discovery and provides evidence of alternative translation products and near-cognate translation initiation events. MOLECULAR & CELLULAR PROTEOMICS. 2013;12(7):1780–90.
MLA
Menschaert, Gerben, Wim Van Criekinge, Tineke Notelaers, et al. “Deep Proteome Coverage Based on Ribosome Profiling Aids Mass Spectrometry-based Protein and Peptide Discovery and Provides Evidence of Alternative Translation Products and Near-cognate Translation Initiation Events.” MOLECULAR & CELLULAR PROTEOMICS 12.7 (2013): 1780–1790. Print.
@article{3209094,
  abstract     = {An increasing number of studies involve integrative analysis of gene and protein expression data, taking advantage of new technologies such as next-generation transcriptome sequencing (RNA-Seq) and highly sensitive mass spectrometry (MS) instrumentation. Recently, a strategy, termed ribosome profiling (or RIBO-seq), based on deep sequencing of ribosome-protected mRNA fragments, indirectly monitoring protein synthesis, has been described. We devised a proteogenomic approach constructing a custom protein sequence search space, built from both SwissProt and RIBO-seq derived translation products, applicable for MS/MS spectrum identification. To record the impact of using the constructed deep proteome database we performed two alternative MS-based proteomic strategies: (I) a regular shotgun proteomic and (II) an N-terminal COFRADIC approach. While the former technique gives an overall assessment on the protein and peptide level, the latter technique, specifically enabling the isolation of N-terminal peptides, is very appropriate in validating the RIBO-seq derived (alternative) translation initiation site profile. We demonstrate that this proteogenomic approach increases the overall protein identification rate with 2.5\% (e.g. new protein products, new protein splice variants, SNP variant proteins, and N-terminally extended forms of known proteins) as compared to only searching UniProtKB-SwissProt. Furthermore, using this custom database, identification of N-terminal COFRADIC data resulted in detection of 16 alternative start sites giving rise to N-terminally extended protein variants besides the identification of four translated uORFs. Notably, the characterization of these new translation products revealed the use of multiple near-cognate (non-AUG) start codons. As deep sequencing techniques are becoming more standard, less expensive, and widespread, we anticipate that mRNA-seq and especially custom-tailored RIBO-seq will become indispensible in the MS-based protein or peptide identification process.},
  author       = {Menschaert, Gerben and Van Criekinge, Wim and Notelaers, Tineke and Koch, Alexander and Crapp{\'e}, Jeroen and Gevaert, Kris and Van Damme, Petra},
  issn         = {1535-9476},
  journal      = {MOLECULAR \& CELLULAR PROTEOMICS},
  keyword      = {upstream translation,ribosome,downstream translation,profiling,start site,near-\-\unmatched{2010}cognate,start codon,RIBO-\-\unmatched{2010}seq,N-\-\unmatched{2010}terminal,COFRADIC,Shotgun proteomics,proteogenomics,OPEN READING FRAMES,FRACTIONAL DIAGONAL CHROMATOGRAPHY,N-TERMINAL PEPTIDES,RNA-SEQ DATA,MESSENGER-RNA,SUBCELLULAR-LOCALIZATION,GENE-EXPRESSION,HUMAN-CELLS,IN-VIVO,GENERATION},
  language     = {eng},
  number       = {7},
  pages        = {1780--1790},
  title        = {Deep proteome coverage based on ribosome profiling aids mass spectrometry-based protein and peptide discovery and provides evidence of alternative translation products and near-cognate translation initiation events},
  url          = {http://dx.doi.org/10.1074/mcp.M113.027540},
  volume       = {12},
  year         = {2013},
}

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