title: Peptidomics for Studying Limited Proteolysis.
abstract: 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.
authors.affiliation: Department of Molecular Pharmacology, National Cerebral and Cardiovascular Center , Osaka 565-8565, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Tsuchiya
authors.affiliation: Department of Molecular Pharmacology, National Cerebral and Cardiovascular Center , Osaka 565-8565, Japan.
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Osaki
authors.affiliation: Department of Molecular Pharmacology, National Cerebral and Cardiovascular Center , Osaka 565-8565, Japan.
authors.forename: Naoto
authors.initials: N
authors.lastname: Minamino
authors.affiliation: Department of Molecular Pharmacology, National Cerebral and Cardiovascular Center , Osaka 565-8565, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Sasaki
mesh_terms: D053607:Adrenomedullin / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000737:chemistry* / Q000378:metabolism; D005347:Fibroblasts / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D061251:Primary Cell Culture; D021382:Protein Sorting Signals / Q000235:genetics; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D040901:Proteomics / Q000379:methods; D051381:Rats; D059467:Transcriptome
chemical_list: D016326:Extracellular Matrix Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D053607:Adrenomedullin
keywords: adrenomedullin; cardiac fibroblasts; ectodomain shedding; endogenous peptide; limited proteolysis; membrane stubs; peptide profiling; peptidomics; regulated intramembrane proteolysis; secretome