In vitro Transcription/Translation System: A Versatile Tool in the Search for Missing Proteins
Author
Summary, in English
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.
Publishing year
2015
Language
English
Pages
3441-3451
Publication/Series
Journal of Proteome Research
Volume
14
Issue
9
Document type
Journal article
Publisher
The American Chemical Society (ACS)
Topic
- Biological Sciences
Keywords
- bioinformatics
- proteomics
- in vitro transcription translation system
- LC-MS
- chromosome centric human proteome project
- missing proteins
Status
Published
ISBN/ISSN/Other
- ISSN: 1535-3893