In vitro transcription/translation system

A versatile tool in the search for missing proteins

Péter Horvatovich, Ákos Végvári, Justin Saul, Jin G. Park, Ji Qiu, Michael Syring, Patrick Pirrotte, Konstantinos Petritis, Tony J. Tegeler, Meraj Aziz, Manuel Fuentes, Paula Diez, Maria Gonzalez-Gonzalez, Nieves Ibarrola, Conrad Droste, Javier De Las Rivas, Concha Gil, Felipe Clemente, Maria Luisa Hernaez, Fernando J. Corrales & 6 others Carol L. Nilsson, Frode S. Berven, Rainer Bischoff, Thomas E. Fehniger, Joshua LaBaer, György Marko-Varga

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Approximately 18% of all human genes purported to encode proteins have not been directly evidenced at the protein level, according to the validation criteria established by neXtProt, and are considered to be "missing" proteins. One of the goals of the Chromosome-Centric Human Proteome Project (C-HPP) is to identify as many of these missing proteins as possible in human samples using mass spectrometry-based methods. To further this goal, a consortium of C-HPP teams (chromosomes 5, 10, 16, and 19) has joined forces to devise new strategies to identify missing proteins by use of a cell-free in vitro transcription/translation system (IVTT). The proposed strategy employs LC-MS/MS data-dependent acquisition (DDA) and targeted selective reaction monitoring (SRM) methods to scrutinize low-complexity samples derived from IVTT. The optimized assays are then applied to identify missing proteins in human cells and tissues. We describe the approach and show proof-of-concept results for development of LC-SRM assays for identification of 18 missing proteins. We believe that the IVTT system, when coupled with downstream mass spectrometric identification, can be applied to identify proteins that have eluded more traditional methods of detection.

Original languageEnglish (US)
Pages (from-to)3441-3451
Number of pages11
JournalJournal of Proteome Research
Volume14
Issue number9
DOIs
StatePublished - Sep 4 2015

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Transcription
Proteins
Chromosomes
Human Chromosomes
Proteome
Assays
Chromosomes, Human, Pair 10
Chromosomes, Human, Pair 5
In Vitro Techniques
Monitoring
Mass spectrometry
Mass Spectrometry
Genes
Cells
Tissue

Keywords

  • bioinformatics
  • Chromosome-Centric Human Proteome Project
  • in vitro transcription/translation system
  • LC-MS
  • Missing proteins
  • proteomics

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

Cite this

Horvatovich, P., Végvári, Á., Saul, J., Park, J. G., Qiu, J., Syring, M., ... Marko-Varga, G. (2015). In vitro transcription/translation system: A versatile tool in the search for missing proteins. Journal of Proteome Research, 14(9), 3441-3451. https://doi.org/10.1021/acs.jproteome.5b00486

In vitro transcription/translation system : A versatile tool in the search for missing proteins. / Horvatovich, Péter; Végvári, Ákos; Saul, Justin; Park, Jin G.; Qiu, Ji; Syring, Michael; Pirrotte, Patrick; Petritis, Konstantinos; Tegeler, Tony J.; Aziz, Meraj; Fuentes, Manuel; Diez, Paula; Gonzalez-Gonzalez, Maria; Ibarrola, Nieves; Droste, Conrad; De Las Rivas, Javier; Gil, Concha; Clemente, Felipe; Hernaez, Maria Luisa; Corrales, Fernando J.; Nilsson, Carol L.; Berven, Frode S.; Bischoff, Rainer; Fehniger, Thomas E.; LaBaer, Joshua; Marko-Varga, György.

In: Journal of Proteome Research, Vol. 14, No. 9, 04.09.2015, p. 3441-3451.

Research output: Contribution to journalArticle

Horvatovich, P, Végvári, Á, Saul, J, Park, JG, Qiu, J, Syring, M, Pirrotte, P, Petritis, K, Tegeler, TJ, Aziz, M, Fuentes, M, Diez, P, Gonzalez-Gonzalez, M, Ibarrola, N, Droste, C, De Las Rivas, J, Gil, C, Clemente, F, Hernaez, ML, Corrales, FJ, Nilsson, CL, Berven, FS, Bischoff, R, Fehniger, TE, LaBaer, J & Marko-Varga, G 2015, 'In vitro transcription/translation system: A versatile tool in the search for missing proteins', Journal of Proteome Research, vol. 14, no. 9, pp. 3441-3451. https://doi.org/10.1021/acs.jproteome.5b00486
Horvatovich, Péter ; Végvári, Ákos ; Saul, Justin ; Park, Jin G. ; Qiu, Ji ; Syring, Michael ; Pirrotte, Patrick ; Petritis, Konstantinos ; Tegeler, Tony J. ; Aziz, Meraj ; Fuentes, Manuel ; Diez, Paula ; Gonzalez-Gonzalez, Maria ; Ibarrola, Nieves ; Droste, Conrad ; De Las Rivas, Javier ; Gil, Concha ; Clemente, Felipe ; Hernaez, Maria Luisa ; Corrales, Fernando J. ; Nilsson, Carol L. ; Berven, Frode S. ; Bischoff, Rainer ; Fehniger, Thomas E. ; LaBaer, Joshua ; Marko-Varga, György. / In vitro transcription/translation system : A versatile tool in the search for missing proteins. In: Journal of Proteome Research. 2015 ; Vol. 14, No. 9. pp. 3441-3451.
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AU - Horvatovich, Péter

AU - Végvári, Ákos

AU - Saul, Justin

AU - Park, Jin G.

AU - Qiu, Ji

AU - Syring, Michael

AU - Pirrotte, Patrick

AU - Petritis, Konstantinos

AU - Tegeler, Tony J.

AU - Aziz, Meraj

AU - Fuentes, Manuel

AU - Diez, Paula

AU - Gonzalez-Gonzalez, Maria

AU - Ibarrola, Nieves

AU - Droste, Conrad

AU - De Las Rivas, Javier

AU - Gil, Concha

AU - Clemente, Felipe

AU - Hernaez, Maria Luisa

AU - Corrales, Fernando J.

AU - Nilsson, Carol L.

AU - Berven, Frode S.

AU - Bischoff, Rainer

AU - Fehniger, Thomas E.

AU - LaBaer, Joshua

AU - Marko-Varga, György

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N2 - Approximately 18% of all human genes purported to encode proteins have not been directly evidenced at the protein level, according to the validation criteria established by neXtProt, and are considered to be "missing" proteins. One of the goals of the Chromosome-Centric Human Proteome Project (C-HPP) is to identify as many of these missing proteins as possible in human samples using mass spectrometry-based methods. To further this goal, a consortium of C-HPP teams (chromosomes 5, 10, 16, and 19) has joined forces to devise new strategies to identify missing proteins by use of a cell-free in vitro transcription/translation system (IVTT). The proposed strategy employs LC-MS/MS data-dependent acquisition (DDA) and targeted selective reaction monitoring (SRM) methods to scrutinize low-complexity samples derived from IVTT. The optimized assays are then applied to identify missing proteins in human cells and tissues. We describe the approach and show proof-of-concept results for development of LC-SRM assays for identification of 18 missing proteins. We believe that the IVTT system, when coupled with downstream mass spectrometric identification, can be applied to identify proteins that have eluded more traditional methods of detection.

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