Quantitative Multiplex Substrate Profiling of Peptidases by Mass Spectrometry.

Quantitative Multiplex Substrate Profiling of Peptidases by Mass Spectrometry. Mol Cell Proteomics. 2019 Jan 31;: Authors: Lapek JD, Jiang Z, Wozniak JM, Arutyunova E, Wang SC, Lemieux MJ, Gonzalez DJ, O'Donoghue AJ Abstract Proteolysis is an integral component of life and has been implicated in many disease processes. To improve our understanding of peptidase function, it is imperative to develop tools to uncover substrate specificity and cleavage efficiency. Here, we combine the quantitative power of tandem mass tags (TMTs) with an established peptide cleavage assay to yield quantitative Multiplex Substrate Profiling by Mass Spectrometry (qMSP-MS). This assay was validated with papain, a well-characterized cysteine peptidase, to generate cleavage efficiency values for hydrolysis of 275 unique peptide bonds in parallel. To demonstrate the breath of this assay, we show that qMSP-MS can uncover the substrate specificity of minimally characterized intramembrane rhomboid peptidases, as well as define hundreds of proteolytic activities in complex biological samples, including secretions from lung cancer cell lines. Importantly, our qMSP-MS library uses synthetic peptides whose termini are unmodified, allowing us to characterize not only endo- but also exo-peptidase activity. Each cleaved peptide sequence can be ranked by turnover rate, and the amino acid sequence of the best substrates can be used for designing fluorescent reporter subst...
Source: Molecular and Cellular Proteomics : MCP - Category: Molecular Biology Authors: Tags: Mol Cell Proteomics Source Type: research