Alternative Strategy To Explore Missing Proteins with Low Molecular Weight

Identifying
more missing proteins (MPs) is an important mission
of C-HPP. With the number of identified MPs being attenuated year
by year (2,949 to 2,129 MPs from 2016 to 2019), we have realized that
the difficulty of exploring the remaining MPs is a challenge in technique.
Herein, we propose a comprehensive strategy to effectively enrich,
separate, and identify proteins with low molecular weights, aiming
at the discovery of MPs. Basically, a protein extract from human placenta
was passed through a C18 SPE column, and the bound proteins that were
eluted were further separated with an SDS-PAGE gel or a 50 kDa cutoff
filter. The separated proteins were subjected to trypsin digestion,
and the MS/MS signals were searched against data sets with two different
digestion modes (full-trypsin and semitrypsin). The strategy was adopted,
resulting in the identification of 4 MPs with 8 unique peptides (≥2
non-nested unique peptides with ≥9 amino acids). Importantly,
the identification of 6 out of 8 of the unique peptides derived from
the MPs was further supported by parallel reaction monitoring, which
confirmed the identification of 3 MPs from human placenta tissues
(Q6NT89: TMF-regulated nuclear protein 1; A0A183: late cornified envelope
protein 6A; and Q6UWQ7: insulin growth factor-like family member 2,
mapped to chromosomes 1, 1, and 19, respectively). The three proteins
ranged in length from 80 aa to 227 aa. The study not only establishes
a feasible strategy for analyzing proteins with low molecular weights
but also fills a small part of a large gap in the list of MPs. The
data obtained in this study are available via ProteomeXchange (PXD014083)
and PeptideAtlas (PASS01389).