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Mass spectrometry and ribosome profiling, a perfect combination towards a more comprehensive identification strategy of true in vivo protein forms

Jeroen Crappé (UGent) , Alexander Koch (UGent) , Sandra Steyaert (UGent) , Wim Van Criekinge (UGent) , Petra Van Damme (UGent) and Gerben Menschaert (UGent)
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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). Recently, a strategy, termed ribosome profiling, based on deep sequencing of ribosome-protected mRNA fragments, indirectly monitoring protein synthesis, has been described. In contrast to routinely employed protein databases in proteomics searches, RIBO-seq derived data gives a more representative expression state and accounts for sequence variation information and alternative translation initiation. To verify the potential of ribosome profiling in providing us with a true snapshot of the translational landscape, we devised a proteogenomic approach generating a database of translation products based on ribosome profiling experiments. The raw and untreated RIBO-seq data is analyzed for both splice isoforms and single nucleotide polymorphisms, as such taking into account transcriptional variation. Next to that, RIBO-seq data for translation start site discovery (treated with harringtonine, lactomidomycin or puromycin) is used to obtain a genome wide blueprint of all possible translation initiation sites and as such taking into account translation variation. By adding protein-DB annotation to the genomic RIBO-seq derived data and after in silico translation a protein database is constructed reflecting the full complexity of the proteome. Using a first version of our proteogenomic approach on an undifferentiated mouse embryonic stem cell line (E14) we could demonstrate an increase of the overall protein identification rate with 2.5% as compared to only searching UniProtKB-SwissProt. Furthermore, 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.

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MLA
Crappé, Jeroen et al. “Mass Spectrometry and Ribosome Profiling, a Perfect Combination Towards a More Comprehensive Identification Strategy of True in Vivo Protein Forms.” Genomics in Europe, Conference Abstracts. 2013. Print.
APA
Crappé, J., Koch, A., Steyaert, S., Van Criekinge, W., Van Damme, P., & Menschaert, G. (2013). Mass spectrometry and ribosome profiling, a perfect combination towards a more comprehensive identification strategy of true in vivo protein forms. Genomics in Europe, Conference abstracts. Presented at the 2013 International conference on Genomics in Europe.
Chicago author-date
Crappé, Jeroen, Alexander Koch, Sandra Steyaert, Wim Van Criekinge, Petra Van Damme, and Gerben Menschaert. 2013. “Mass Spectrometry and Ribosome Profiling, a Perfect Combination Towards a More Comprehensive Identification Strategy of True in Vivo Protein Forms.” In Genomics in Europe, Conference Abstracts.
Chicago author-date (all authors)
Crappé, Jeroen, Alexander Koch, Sandra Steyaert, Wim Van Criekinge, Petra Van Damme, and Gerben Menschaert. 2013. “Mass Spectrometry and Ribosome Profiling, a Perfect Combination Towards a More Comprehensive Identification Strategy of True in Vivo Protein Forms.” In Genomics in Europe, Conference Abstracts.
Vancouver
1.
Crappé J, Koch A, Steyaert S, Van Criekinge W, Van Damme P, Menschaert G. Mass spectrometry and ribosome profiling, a perfect combination towards a more comprehensive identification strategy of true in vivo protein forms. Genomics in Europe, Conference abstracts. 2013.
IEEE
[1]
J. Crappé, A. Koch, S. Steyaert, W. Van Criekinge, P. Van Damme, and G. Menschaert, “Mass spectrometry and ribosome profiling, a perfect combination towards a more comprehensive identification strategy of true in vivo protein forms,” in Genomics in Europe, Conference abstracts, Ghent, Belgium, 2013.
@inproceedings{4197342,
  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). Recently, a strategy, termed ribosome profiling, based on deep sequencing of ribosome-protected mRNA fragments, indirectly monitoring protein synthesis, has been described. In contrast to routinely employed protein databases in proteomics searches, RIBO-seq derived data gives a more representative expression state and accounts for sequence variation information and alternative translation initiation. 
To verify the potential of ribosome profiling in providing us with a true snapshot of the translational landscape, we devised a proteogenomic approach generating a database of translation products based on ribosome profiling experiments. The raw and untreated RIBO-seq data is analyzed for both splice isoforms and single nucleotide polymorphisms, as such taking into account transcriptional variation. Next to that, RIBO-seq data for translation start site discovery (treated with harringtonine, lactomidomycin or puromycin) is used to obtain a genome wide blueprint of all possible translation initiation sites and as such taking into account translation variation. By adding protein-DB annotation to the genomic RIBO-seq derived data and after in silico translation a protein database is constructed reflecting the full complexity of the proteome. 
Using a first version of our proteogenomic approach on an undifferentiated mouse embryonic stem cell line (E14) we could demonstrate an increase of the overall protein identification rate with 2.5% as compared to only searching UniProtKB-SwissProt. Furthermore, 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.},
  author       = {Crappé, Jeroen and Koch, Alexander and Steyaert, Sandra and Van Criekinge, Wim and Van Damme, Petra and Menschaert, Gerben},
  booktitle    = {Genomics in Europe, Conference abstracts},
  language     = {eng},
  location     = {Ghent, Belgium},
  title        = {Mass spectrometry and ribosome profiling, a perfect combination towards a more comprehensive identification strategy of true in vivo protein forms},
  year         = {2013},
}