title: LC-MS based cleavage site profiling of the proteases ADAM10 and ADAM17 using proteome-derived peptide libraries.
abstract: A Disintegrin and Metalloproteinase 10 (ADAM10) and ADAM17 catalyze ectodomain shedding of a number of cell surface proteins important for embryonic development and tissue homeostasis. Changes in the expression levels or dysregulated proteolytic activity of ADAM10 and ADAM17 have been shown to play important roles in multiple diseases such as inflammation, cancer, and neurodegenerative disorders. Despite the well documented substrate repertoire of ADAM10 and ADAM17, little is known about their cleavage site specificity. We optimized Q-PICS (Quantitative Proteomics for the Identification of Cleavage Sites) to elucidate the cleavage site specificity of recombinant murine ADAM10 and ADAM17. Two different yeast proteome-derived peptide libraries were used and samples were analyzed by LC-MALDI and LC-ESI MS in parallel. We show that the largest difference in the cleavage site specificities of ADAM10 and ADAM17 is at the P1' site: while both enzymes cleave N-terminal of leucine, only ADAM10 shows additional preference toward aromatic amino acids, whereas ADAM17 exhibits the highest preference for valine. Together with further amino acid preferences more adjacent to the scissile bond, our data is in good agreement with ADAM10/17 cleavage sites previously identified in native substrates. Overall, the precise identification of ADAM10 and ADAM17 cleavage site specificity provides the basis for better substrate identification in vivo and the generation of specific inhibitors or activity based probes.
authors.affiliation: AG Systematische Proteomforschung, Institut für Experimentelle Medizin, Christian-Albrechts-Universität zu Kiel , Niemannsweg 11, 24105 Kiel, Germany.
authors.forename: Joanna
authors.initials: J
authors.lastname: Tucher
authors.forename: Dennis
authors.initials: D
authors.lastname: Linke
authors.forename: Tomas
authors.initials: T
authors.lastname: Koudelka
authors.forename: Liam
authors.initials: L
authors.lastname: Cassidy
authors.forename: Claudia
authors.initials: C
authors.lastname: Tredup
authors.forename: Rielana
authors.initials: R
authors.lastname: Wichert
authors.forename: Claus
authors.initials: C
authors.lastname: Pietrzik
authors.forename: Christoph
authors.initials: C
authors.lastname: Becker-Pauly
authors.forename: Andreas
authors.initials: A
authors.lastname: Tholey
mesh_terms: D051722:ADAM Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D000072197:ADAM10 Protein; D000072198:ADAM17 Protein; D053829:Amyloid Precursor Protein Secretases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002853:Chromatography, Liquid; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D019151:Peptide Library; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
chemical_list: D008565:Membrane Proteins; D010446:Peptide Fragments; D019151:Peptide Library; D020543:Proteome; D053829:Amyloid Precursor Protein Secretases; D051722:ADAM Proteins; D000072197:ADAM10 Protein; C491234:Adam10 protein, mouse; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; C000606463:Adam17 protein, mouse