title: In-situ generation of differential sensors that fingerprint kinases and the cellular response to their expression.
abstract: Mitogen-activated protein (MAP) kinases are responsible for many cellular functions, and their malfunction manifests itself in several human diseases. Usually, monitoring the phosphorylation states of MAP kinases in vitro requires the preparation and purification of the proteins or Western blotting. Herein, we report an array sensing approach for the differentiation of MAP kinases and their phosphorylated counterparts in vitro. This technique utilizes a library of differential receptors created in situ containing peptides known for affinity to MAP kinases, and a Zn(II)-dipicolylamine complex that binds phosphate groups on proteins. An indicator-displacement assay signals the binding of the individual receptors to the kinases, while chemometrics is used to create a fingerprint for the kinases and their state of activity. For example, linear discriminant analysis correctly identified kinase activity with a classification accuracy of 97.5% in vitro, while the cellular response to kinase expression was classified with 100% accuracy.
authors.affiliation: Department of Chemistry and Biochemistry and ‡Division of Medicinal Chemistry, The University of Texas at Austin , Austin, Texas 78712, United States.
authors.forename: Diana
authors.initials: D
authors.lastname: Zamora-Olivares
authors.forename: Tamer S
authors.initials: TS
authors.lastname: Kaoud
authors.forename: Kevin N
authors.initials: KN
authors.lastname: Dalby
authors.forename: Eric V
authors.initials: EV
authors.lastname: Anslyn
mesh_terms: D000595:Amino Acid Sequence; D015374:Biosensing Techniques; D004789:Enzyme Activation; D057075:Enzyme Assays; D057809:HEK293 Cells; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism
chemical_list: D010455:Peptides; D020033:Protein Isoforms; D020928:Mitogen-Activated Protein Kinases