title: A bead-based western for high-throughput cellular signal transduction analyses.
abstract: Dissecting cellular signalling requires the analysis of large number of proteins. The DigiWest approach we describe here transfers the western blot to a bead-based microarray platform. By combining gel-based protein separation with immobilization on microspheres, hundreds of replicas of the initial blot are created, thus enabling the comprehensive analysis of limited material, such as cells collected by laser capture microdissection, and extending traditional western blotting to reach proteomic scales. The combination of molecular weight resolution, sensitivity and signal linearity on an automated platform enables the rapid quantification of hundreds of specific proteins and protein modifications in complex samples. This high-throughput western blot approach allowed us to identify and characterize alterations in cellular signal transduction that occur during the development of resistance to the kinase inhibitor Lapatinib, revealing major changes in the activation state of Ephrin-mediated signalling and a central role for p53-controlled processes.
authors.affiliation: NMI Natural and Medical Sciences Institute at the University of Tübingen, 72770 Reutlingen, Germany.|Pharmaceutical Biotechnology, Eberhard-Karls-Universität Tübingen, Tübingen, 72770 Reutlingen, Germany.
authors.forename: Fridolin
authors.initials: F
authors.lastname: Treindl
authors.affiliation: Chair for Proteomics and Bioanalytics, Center of Life and Food Sciences Weihenstephan, Technische Universität München, 85354 Freising, Germany.|Center for Integrated Protein Science Munich, 85354 Freising, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Ruprecht
authors.affiliation: NMI Natural and Medical Sciences Institute at the University of Tübingen, 72770 Reutlingen, Germany.|Pharmaceutical Biotechnology, Eberhard-Karls-Universität Tübingen, Tübingen, 72770 Reutlingen, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Beiter
authors.affiliation: Department of Obstetrics and Gynecology, Medical Faculty and University Hospital of the Heinrich-Heine University Duesseldorf, 40225 Düsseldorf, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Schultz
authors.affiliation: NMI Natural and Medical Sciences Institute at the University of Tübingen, 72770 Reutlingen, Germany.
authors.forename: Anette
authors.initials: A
authors.lastname: Döttinger
authors.affiliation: Department of Pathology, University Hospital Tübingen, 72076 Tübingen, Germany.
authors.forename: Annette
authors.initials: A
authors.lastname: Staebler
authors.affiliation: NMI Natural and Medical Sciences Institute at the University of Tübingen, 72770 Reutlingen, Germany.
authors.forename: Thomas O
authors.initials: TO
authors.lastname: Joos
authors.affiliation: NMI Natural and Medical Sciences Institute at the University of Tübingen, 72770 Reutlingen, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Kling
authors.affiliation: NMI Natural and Medical Sciences Institute at the University of Tübingen, 72770 Reutlingen, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Poetz
authors.affiliation: Department of Obstetrics and Gynecology, Medical Faculty and University Hospital of the Heinrich-Heine University Duesseldorf, 40225 Düsseldorf, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Fehm
authors.affiliation: Department of Obstetrics and Gynecology, Medical Faculty and University Hospital of the Heinrich-Heine University Duesseldorf, 40225 Düsseldorf, Germany.
authors.forename: Hans
authors.initials: H
authors.lastname: Neubauer
authors.affiliation: Chair for Proteomics and Bioanalytics, Center of Life and Food Sciences Weihenstephan, Technische Universität München, 85354 Freising, Germany.|Center for Integrated Protein Science Munich, 85354 Freising, Germany.|Bavarian Biomolecular Mass Spectrometry Center (BayBioMS), Technische Universität München, 85354 Freising, Germany.
authors.forename: Bernhard
authors.identifier: http://orcid.org/0000-0002-9094-1677
authors.initials: B
authors.lastname: Kuster
authors.affiliation: NMI Natural and Medical Sciences Institute at the University of Tübingen, 72770 Reutlingen, Germany.|Pharmaceutical Biotechnology, Eberhard-Karls-Universität Tübingen, Tübingen, 72770 Reutlingen, Germany.
authors.forename: Markus F
authors.initials: MF
authors.lastname: Templin