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
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.