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J Proteome Res. 2015 Sep 4;14(9):3441-51. doi: 10.1021/acs.jproteome.5b00486. Epub 2015 Jul 28.

In Vitro Transcription/Translation System: A Versatile Tool in the Search for Missing Proteins.

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Analytical Biochemistry, Department of Pharmacy, University of Groningen , A. Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Department of Pharmacology & Toxicology, The University of Texas Medical Branch , 301 University Boulevard, Galveston, Texas 77555-1074, United States.
Center for Personalized Diagnostics, Biodesign Institute, Arizona State University , Tempe, Arizona 85287, United States.
Center for Proteomics, Translational Genomics Research Institute , Phoenix, Arizona 85004, United States.
Pathology Research, Phoenix Children's Hospital , 1919 East Thomas Road, Phoenix, Arizona 85016, United States.
Department of Microbiology & Proteomics Unit, University Complutense , 28040 Madrid, Spain.
Center for Applied Medical Research (CIMA), University of Navarra, PRB2-ProteoRed-ISCIII, IDISNA, Ciberhed , 31008 Pamplona, Spain.
Proteomics Unit (PROBE), Department of Biomedicine, University of Bergen , Postbox 7804, N-5009 Bergen, Norway.
The Norwegian Multiple Sclerosis Competence Centre, Department of Neurology, Haukeland University Hospital , Postbox 1400, 5021 Bergen, Norway.
First Department of Surgery, Tokyo Medical University , 6-7-1 Nishishinjuku Shinjuku-ku, 160-0023 Tokyo, Japan.


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.


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

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