In vitro transcription/translation system: A versatile tool in the search for missing proteins

et al.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.Support from the Cancer Prevention Research Institute of Texas (RML 1122 to C.L.N.) and the University of Texas Medical Branch (C.L.N.) is gratefully acknowledged. Financial support from the Carlos III Health Institute of Spain (ISCIII, FIS PI11/02114/FIS PI14/01538 and FIS PI12/00624), Fondos FEDER (EU) is gratefully acknowledged. The proteomics units at CIC and UCM belong to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001. P.D. and C.D. are supported by JCYL-EDU/346/2013 Ph.D. scholarships.Peer Reviewe

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

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 as “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 translation. 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 eighteen “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