Limited
proteolysis is a pivotal mechanism regulating protein functions.
Identifying physiologically or pathophysiologically relevant cleavage
sites helps to develop molecular tools that can be used for diagnostics
or therapeutics. During proteolysis of secretory and membrane proteins,
part of the cleaved protein is liberated and destined to undergo degradation
but should retain original cleavage sites created by proteolytic enzymes.
We profiled endogenous peptides accumulated for 4 h in media conditioned
by primary cultured rat cardiac fibroblasts. A total of 3916 redundant
peptide sequences from 94 secretory proteins and membrane proteins
served to identify limited cleavage sites, both annotated and unannotated,
for signal peptide or propeptide removal, peptide hormone processing,
ectodomain shedding, and regulated intramembrane proteolysis. Incorrectly
predicted signal cleavage sites are found in typical proteins such
as extracellular matrix proteins and the peptide hormone precursor
adrenomedullin ADM. The revealed signal peptide cleavage site for
ADM was experimentally verified by identifying the major molecular
form of flanking proadrenomedullin N-terminal peptide. We suggest
that profiling of endogenous peptides, like transcriptome sequence
reads, makes sense in regular cells such as fibroblasts and that peptidomics
provides insight into proteolysis-regulated protein functions.