title: SPRi-MALDI MS: characterization and identification of a kinase from cell lysate by specific interaction with different designed ankyrin repeat proteins.
abstract: We report on the direct coupling of surface plasmon resonance imaging (SPRi) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) for the investigation of specific, non-covalent interactions, using the example of designed ankyrin repeat proteins (DARPins) and ribosomal protein S6 kinase 2 (RPS6KA2) directly from lysate of SH-SY5Y cells, derived from human bone marrow. Due to an array format, tracing of binding kinetics of numerous DARPins simultaneously and in real time becomes possible. By optimizing both the proteolytic digest directly on the SPRi chip (amount of trypsin, incubation time, and temperature) as well as the MALDI matrix application (concentration of matrix and number of spray cycles), we are able to identify the specific interaction with RPS6KA2 directly from the cell lysate at a surface coverage of only 0.8 fmol/mm2. Graphical Abstract Workflow of the direct coupling of SPRi with MALDI mass spectrometry.
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zurich, Vladimir-Prelog-Weg 3, 8093, Zurich, Switzerland.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Anders
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
authors.forename: Jonas V
authors.initials: JV
authors.lastname: Schaefer
authors.affiliation: Horiba Jobin Yvon S.A.S., Avenue de la Vauve-Passage Jobin Yvon, CS 45002, 91120, Palaiseau, France.
authors.forename: Fatima-Ezzahra
authors.initials: FE
authors.lastname: Hibti
authors.affiliation: Horiba Jobin Yvon S.A.S., Avenue de la Vauve-Passage Jobin Yvon, CS 45002, 91120, Palaiseau, France.
authors.forename: Chiraz
authors.initials: C
authors.lastname: Frydman
authors.affiliation: Bruker Daltonics, Fahrenheitstr. 4, 28359, Bremen, Germany.
authors.forename: Detlev
authors.initials: D
authors.lastname: Suckau
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zurich, Vladimir-Prelog-Weg 3, 8093, Zurich, Switzerland. zenobi@org.chem.ethz.ch.
authors.forename: Renato
authors.initials: R
authors.lastname: Zenobi
mesh_terms: D017089:Ankyrin Repeat; D006801:Humans; D010770:Phosphotransferases / Q000737:chemistry*; D059748:Proteolysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D010770:Phosphotransferases
keywords: Cell lysate; Designed ankyrin repeat proteins (DAPRins); Label-free; MALDI mass spectrometry; Surface plasmon resonance imaging (SPRi)