期刊
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
卷 409, 期 7, 页码 1827-1836出版社
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-016-0127-3
关键词
Surface plasmon resonance imaging (SPRi); MALDI mass spectrometry; Designed ankyrin repeat proteins (DAPRins); Cell lysate; Label-free
资金
- SPRi
- Bruker Daltonics
- Horiba Jobin Yvon
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).
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