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

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