Peptide- and Protein-Level Combined Strategy for Analyzing Newly Synthesized Proteins by Integrating Tandem Orthogonal Proteolysis with Cleavable Bioorthogonal Tagging

A newly synthesized proteome reflects
perturbations sensitively
and maintains homeostasis in cells. To investigate the low abundant
newly synthesized proteins (NSPs) from a complex background proteome,
an enrichment process with high selectivity and reliability is essential.
Here, we have developed a strategy to realize comprehensive analysis
of NSPs by integrating tandem orthogonal proteolysis (TOP) with cleavable
bioorthogonal tagging (CBOT) called TOP–CBOT. A solid-phase-conjugated
probe with a clickable moiety and a protease-cleavable site was designed, which allowed NSPs to be
covalently captured along with tandem release by trypsin and orthogonal
tobacco etch virus (TEV) protease. Our method has integrated the advantages
of protein-level and peptide-level enrichment. Trypsin digests larger
number of peptides from the recovered proteins for NSPs identification
and quantification, while the specific tag-contained peptides from
TEV data set enabled further NSPs confirmation. Integrating information
from two complementary data sets, the reliability in NSPs identification
and quantitation were remarkably enhanced. A total of 3699 proteins
were recovered in the trypsin data set. Additionally, 1931 proteins
were confirmed as NSPs with 5019 identified peptides in the TEV data
set, over 90% of which were overlapped with the tryptic data set.
Our strategy was further applied to profile NSP degradation kinetics
during rapamycin-induced macroautophagy. The newly synthesized proteome
displayed varied alteration of degradation rates among stimulation
and more than half of NSPs showed decreased half-lives during autophagy.