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SPRi-MALDI MS: characterization and identification of a kinase from cell lysate by specific interaction with different designed ankyrin repeat proteins


Anders, Ulrike; Schaefer, Jonas V; Hibti, Fatima-Ezzahra; Frydman, Chiraz; Suckau, Detlev; Plückthun, Andreas; Zenobi, Renato (2017). SPRi-MALDI MS: characterization and identification of a kinase from cell lysate by specific interaction with different designed ankyrin repeat proteins. Analytical and Bioanalytical Chemistry, 409(7):1827-1836.

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/mm(2). Graphical Abstract Workflow of the direct coupling of SPRi with MALDI mass spectrometry.

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/mm(2). Graphical Abstract Workflow of the direct coupling of SPRi with MALDI mass spectrometry.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:March 2017
Deposited On:23 Jun 2017 09:52
Last Modified:24 Jun 2017 07:53
Publisher:Springer
ISSN:1618-2642
Publisher DOI:https://doi.org/10.1007/s00216-016-0127-3
PubMed ID:27987025

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