title: Ampicillin/penicillin-binding protein interactions as a model drug-target system to optimize affinity pull-down and mass spectrometric strategies for target and pathway identification.
abstract: The identification and validation of the targets of active compounds identified in cell-based assays is an important step in preclinical drug development. New analytical approaches that combine drug affinity pull-down assays with mass spectrometry (MS) could lead to the identification of new targets and druggable pathways. In this work, we investigate a drug-target system consisting of ampicillin- and penicillin-binding proteins (PBPs) to evaluate and compare different amino-reactive resins for the immobilization of the affinity compound and mass spectrometric methods to identify proteins from drug affinity pull-down assays. First, ampicillin was immobilized onto various amino-reactive resins, which were compared in the ampicillin-PBP model with respect to their nonspecific binding of proteins from an Escherichia coli membrane extract. Dynal M-270 magnetic beads were chosen to further study the system as a model for capturing and identifying the targets of ampicillin, PBPs that were specifically and covalently bound to the immobilized ampicillin. The PBPs were identified, after in situ digestion of proteins bound to ampicillin directly on the beads, by using either one-dimensional (1-D) or two-dimensional (2-D) liquid chromatography (LC) separation techniques followed by tandem mass spectrometry (MS/MS) analysis. Alternatively, an elution with N-lauroylsarcosine (sarcosyl) from the ampicillin beads followed by in situ digestion and 2-D LC-MS/MS analysis identified proteins potentially interacting noncovalently with the PBPs or the ampicillin. The in situ approach required only little time, resources, and sample for the analysis. The combination of drug affinity pull-down assays with in situ digestion and 2-D LC-MS/MS analysis is a useful tool in obtaining complex information about a primary drug target as well as its protein interactors.
authors.affiliation: Prolexys Pharmaceuticals Inc, Salt Lake City, UT 84116, USA. mvonrech@prolexys.com
authors.forename: Moritz
authors.initials: M
authors.lastname: von Rechenberg
authors.forename: Brian Kelly
authors.initials: BK
authors.lastname: Blake
authors.forename: Yew-Seng J
authors.initials: YS
authors.lastname: Ho
authors.forename: Yuejun
authors.initials: Y
authors.lastname: Zhen
authors.forename: Cindy Lou
authors.initials: CL
authors.lastname: Chepanoske
authors.forename: Bonnie E
authors.initials: BE
authors.lastname: Richardson
authors.forename: Nafei
authors.initials: N
authors.lastname: Xu
authors.forename: Vladimir
authors.initials: V
authors.lastname: Kery
mesh_terms: D000667:Ampicillin / Q000378:metabolism*; D016503:Drug Delivery Systems; D013058:Mass Spectrometry / Q000379:methods; D008863:Microspheres; D046915:Penicillin-Binding Proteins / Q000378:metabolism*; D012521:Sarcosine / Q000031:analogs & derivatives / Q000737:chemistry; D012685:Sepharose / Q000031:analogs & derivatives
chemical_list: D046915:Penicillin-Binding Proteins; C032392:Affi-Gel 10; C025231:sarkosyl; D000667:Ampicillin; D012685:Sepharose; D012521:Sarcosine