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An extra dimension in protein tagging by quantifying universal proteotypic peptides using targeted proteomics

Giel Vandemoortele (UGent) , An Staes (UGent) , Giulia Gonnelli (UGent) , Noortje Samyn (UGent) , Delphine De Sutter (UGent) , Elien Vandermarliere (UGent) , Evy Timmerman (UGent) , Kris Gevaert (UGent) , Lennart Martens (UGent) and Sven Eyckerman (UGent)
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Abstract
The use of protein tagging to facilitate detailed characterization of target proteins has not only revolutionized cell biology, but also enabled biochemical analysis through efficient recovery of the protein complexes wherein the tagged proteins reside. The endogenous use of these tags for detailed protein characterization is widespread in lower organisms that allow for efficient homologous recombination. With the recent advances in genome engineering, tagging of endogenous proteins is now within reach for most experimental systems, including mammalian cell lines cultures. In this work, we describe the selection of peptides with ideal mass spectrometry characteristics for use in quantification of tagged proteins using targeted proteomics. We mined the proteome of the hyperthermophile Pyrococcus furiosus to obtain two peptides that are unique in the proteomes of all known model organisms (proteotypic) and allow sensitive quantification of target proteins in a complex background. By combining these 'Proteotypic peptides for Quantification by SRM' (PQS peptides) with epitope tags, we demonstrate their use in co-immunoprecipitation experiments upon transfection of protein pairs, or after introduction of these tags in the endogenous proteins through genome engineering. Endogenous protein tagging for absolute quantification provides a powerful extra dimension to protein analysis, allowing the detailed characterization of endogenous proteins.
Keywords
IDENTIFICATION, PURIFICATION, CANCER, SYSTEM, QUANTIFICATION, FUSION PROTEINS, P53-MDM2 INTERACTION, ANTIPEPTIDE ANTIBODIES, QUANTITATIVE PROTEOMICS, MICROARRAYS

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Chicago
Vandemoortele, Giel, An Staes, Giulia Gonnelli, Noortje Samyn, Delphine De Sutter, Elien Vandermarliere, Evy Timmerman, Kris Gevaert, Lennart Martens, and Sven Eyckerman. 2016. “An Extra Dimension in Protein Tagging by Quantifying Universal Proteotypic Peptides Using Targeted Proteomics.” Scientific Reports 6.
APA
Vandemoortele, G., Staes, A., Gonnelli, G., Samyn, N., De Sutter, D., Vandermarliere, E., Timmerman, E., et al. (2016). An extra dimension in protein tagging by quantifying universal proteotypic peptides using targeted proteomics. SCIENTIFIC REPORTS, 6.
Vancouver
1.
Vandemoortele G, Staes A, Gonnelli G, Samyn N, De Sutter D, Vandermarliere E, et al. An extra dimension in protein tagging by quantifying universal proteotypic peptides using targeted proteomics. SCIENTIFIC REPORTS. 2016;6.
MLA
Vandemoortele, Giel, An Staes, Giulia Gonnelli, et al. “An Extra Dimension in Protein Tagging by Quantifying Universal Proteotypic Peptides Using Targeted Proteomics.” SCIENTIFIC REPORTS 6 (2016): n. pag. Print.
@article{7286625,
  abstract     = {The use of protein tagging to facilitate detailed characterization of target proteins has not only revolutionized cell biology, but also enabled biochemical analysis through efficient recovery of the protein complexes wherein the tagged proteins reside. The endogenous use of these tags for detailed protein characterization is widespread in lower organisms that allow for efficient homologous recombination. With the recent advances in genome engineering, tagging of endogenous proteins is now within reach for most experimental systems, including mammalian cell lines cultures. In this work, we describe the selection of peptides with ideal mass spectrometry characteristics for use in quantification of tagged proteins using targeted proteomics. We mined the proteome of the hyperthermophile Pyrococcus furiosus to obtain two peptides that are unique in the proteomes of all known model organisms (proteotypic) and allow sensitive quantification of target proteins in a complex background. By combining these 'Proteotypic peptides for Quantification by SRM' (PQS peptides) with epitope tags, we demonstrate their use in co-immunoprecipitation experiments upon transfection of protein pairs, or after introduction of these tags in the endogenous proteins through genome engineering. Endogenous protein tagging for absolute quantification provides a powerful extra dimension to protein analysis, allowing the detailed characterization of endogenous proteins.},
  articleno    = {27220},
  author       = {Vandemoortele, Giel and Staes, An and Gonnelli, Giulia and Samyn, Noortje and De Sutter, Delphine and Vandermarliere, Elien and Timmerman, Evy and Gevaert, Kris and Martens, Lennart and Eyckerman, Sven},
  issn         = {2045-2322},
  journal      = {SCIENTIFIC REPORTS},
  keyword      = {IDENTIFICATION,PURIFICATION,CANCER,SYSTEM,QUANTIFICATION,FUSION PROTEINS,P53-MDM2 INTERACTION,ANTIPEPTIDE ANTIBODIES,QUANTITATIVE PROTEOMICS,MICROARRAYS},
  language     = {eng},
  pages        = {12},
  title        = {An extra dimension in protein tagging by quantifying universal proteotypic peptides using targeted proteomics},
  url          = {http://dx.doi.org/10.1038/srep27220},
  volume       = {6},
  year         = {2016},
}

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