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

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• 作者：Ulrike Anders ; Jonas V. Schaefer…
• 关键词：Surface plasmon resonance imaging (SPRi) ; MALDI mass spectrometry ; Designed ankyrin repeat proteins (DAPRins) ; Cell lysate ; Label ; free
• 刊名：Analytical and Bioanalytical Chemistry
• 出版年：2017
• 出版时间：March 2017
• 年：2017
• 卷：409
• 期：7
• 页码：1827-1836
• 全文大小：
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Analytical Chemistry; Biochemistry, general; Laboratory Medicine; Characterization and Evaluation of Materials; Food Science; Monitoring/Environmental Analysis;
• 出版者：Springer Berlin Heidelberg
• ISSN：1618-2650
• 卷排序：409

文摘

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.