Screening of Missing Proteins in the Human Liver Proteome by Improved MRM-Approach-Based Targeted Proteomics

To completely annotate the human
genome, the task of identifying
and characterizing proteins that currently lack mass spectrometry
(MS) evidence is inevitable and urgent. In this study, as the first
effort to screen missing proteins in large scale, we developed an
approach based on SDS-PAGE followed by liquid chromatography–multiple
reaction monitoring (LC-MRM), for screening of those missing proteins
with only a single peptide hit in the previous liver proteome data
set. Proteins extracted from normal human liver were separated in
SDS-PAGE and digested in split gel slice, and the resulting digests
were then subjected to LC-schedule MRM analysis. The MRM assays were
developed through synthesized crude peptides for target peptides.
In total, the expressions of 57 target proteins were confirmed from
185 MRM assays in normal human liver tissues. Among the proved 57
one-hit wonders, 50 proteins are of the minimally redundant set in
the PeptideAtlas database, 7 proteins even have none MS-based information
previously in various biological processes. We conclude that our SDS-PAGE-MRM
workflow can be a powerful approach to screen missing or poorly characterized
proteins in different samples and to provide their quantity if detected.
The MRM raw data have been uploaded to ISB/SRM Atlas/PASSEL (PXD000648).