Broad Coverage Identification of Multiple Proteolytic Cleavage Site Sequences in Complex High Molecular Weight Proteins Using Quantitative Proteomics as a Complement to Edman Sequencing

摘要: Proteolytic processing modifies the pleiotropic functions of many large, complex, and modular proteins can generate cleavage products with new biological activity. The identification exact proteolytic sites in extracellular matrix laminins, fibronectin, other is not only important for understanding protein turnover but needed bioactive products. Several such have recently been recognized that are suggested to play cellular regulatory roles processes, including angiogenesis. However, identifying multiple large challenging as N-terminal Edman sequencing often closely spaced fragments on SDS-PAGE gels difficult, thus limiting throughput coverage. We developed a liquid chromatography-mass spectrometry approach we call amino-terminal oriented mass substrates (ATOMS) solution. ATOMS utilizes efficient low cost dimethylation isotopic labeling original proteolytically generated N termini followed by quantitative tandem analysis. Being peptide-centric approach, dependent resolution limits similar mass. demonstrate reliably identifies per reaction complex proteins. Fifty-five neutrophil elastase were identified laminin-1 fibronectin-1 34 more metalloproteinase cleavage. Hence, our degradomics offers complimentary alternative broad applicability high molecular weight after vitro assays. therefore be useful cleaved proteases pathology bioactivity screening.