Detection of Missing Proteins Using the PRIDE Database as a Source of Mass Spectrometry Evidence

The current catalogue of the human
proteome is not yet complete,
as experimental proteomics evidence is still elusive for a group of
proteins known as the missing proteins. The Human Proteome Project
(HPP) has been successfully using technology and bioinformatic resources
to improve the characterization of such challenging proteins. In this
manuscript, we propose a pipeline starting with the mining of the
PRIDE database to select a group of data sets potentially enriched
in missing proteins that are subsequently analyzed for protein identification
with a method based on the statistical analysis of proteotypic peptides.
Spermatozoa and the HEK293 cell line were found to be a promising
source of missing proteins and clearly merit further attention in
future studies. After the analysis of the selected samples, we found
342 PSMs, suggesting the presence of 97 missing proteins in human
spermatozoa or the HEK293 cell line, while only 36 missing proteins
were potentially detected in the retina, frontal cortex, aorta thoracica,
or placenta. The functional analysis of the missing proteins detected
confirmed their tissue specificity, and the validation of a selected
set of peptides using targeted proteomics (SRM/MRM assays) further
supports the utility of the proposed pipeline. As illustrative examples,
DNAH3 and TEPP in spermatozoa, and UNCX and ATAD3C in HEK293 cells
were some of the more robust and remarkable identifications in this
study. We provide evidence indicating the relevance to carefully analyze
the ever-increasing MS/MS data available from PRIDE and other repositories
as sources for missing proteins detection in specific biological matrices
as revealed for HEK293 cells.