Open-pFind Verified Four Missing Proteins from Multi-Tissues

The Chromosome-Centric Human Proteome
Project (C-HPP) was launched
in 2012 to perfect the annotation of human protein existence by identifying
stronger evidence of the expression of missing proteins (MPs) at the
protein level. After an 8 year effort all over the world, the number
of MPs in the neXtProt database significantly decreased from 5511
(2012-02-24) to 1899 (2020-01-17). It is now more difficult to provide
confident evidence of the remaining MPs because of their specific
characteristics, including low abundance, low molecular weight, unexpected
modifications, transmembrane structure, tissue-expression specificity,
and so on. A higher resolution mass spectrometry (MS) interpretation
engine might provide an opportunity to identify these buried MPs in
complex samples by the combination with multi-tissue large-scale proteomics.
In this study, open-pFind was used to dig MPs from 20 pairs of healthy
human tissues by Wang et al. (Mol. Syst. Biol. 2019, 15 (2), e8503) combined with our large-scale testis data
set digested by three enzymes (Glu-C, Lys-C, and trypsin) with specificity
for different amino acid residues (J. Proteme Res. 2019, 18 (12), 4189−4196). A total of 1 535 536
peptides with 17 283 477 peptide-spectrum matches (PSMs)
were mapped to 14 279 protein entries at a false discovery
rate of <1% at the PSM, peptide, and protein levels. A total of
103 MP candidates were identified, among which 86 candidates had more
unique peptide numbers compared with our single testis tissue. After
rigorous screening, manual checks, peptide synthesis, and matching
with documented peptides from PeptideAtlas, we validated four MPs,
P0C7T8 (duodenum and small intestine), Q8WWZ4 (stomach and rectum),
Q8IV35 (fallopian tube), and O14921 (tonsil), at the protein level.
All MS raw files have been deposited to the ProteomeXchange with identifier
PXD021391.