Analysis of protein tyrosine phosphatase interactions with microarrayed phosphopeptide substrates using imaging mass spectrometry.
Analysis of protein tyrosine phosphatase interactions with microarrayed phosphopeptide substrates using imaging mass spectrometry.
复制标题
使用成像质谱分析蛋白质酪氨酸磷酸酶与微阵列磷酸肽底物的相互作用。
DOI：
10.1016/j.ab.2013.07.031
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发表时间：
2013
影响因子：
2.9
通讯作者：
Ulrich,RobertG
中科院分区：
文献类型：
--
作者：
McKee,ChristopherJ;Hines,HarryB;Ulrich,RobertG
肽和重组蛋白质文库的微阵列通常用于蛋白质-蛋白质相互作用和酶活性的高通量研究。成像质谱法（IMS）目前被用作一种方法，以定位薄组织切片和其他表面上的分析物。在这里，我们应用IMS作为一种无标记的手段来分析蛋白质-肽相互作用的基于微阵列的磷酸酶测定。该IMS策略通过在芯片表面上收集预定义的基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱光谱光栅，在一个复合图像中可视化整个微阵列。检查细菌酪氨酸磷酸酶YopH，我们使用IMS作为一种无标记的手段来可视化酶的结合和活性与微阵列磷酸肽库印刷在芯片上涂有金或铟锡氧化物。此外，我们表明，基于微阵列的IMS可以与表面等离子体共振成像加上动力学分析测量结合相互作用。这里描述的方法是在许多现代MALDI-TOF仪器的能力，并具有通用性的微阵列分析的无标记分析。
Microarrays of peptide and recombinant protein libraries are routinely used for high-throughput studies of protein–protein interactions and enzymatic activities. Imaging mass spectrometry (IMS) is currently applied as a method to localize analytes on thin tissue sections and other surfaces. Here, we have applied IMS as a label-free means to analyze protein–peptide interactions in a microarray-based phosphatase assay. This IMS strategy visualizes the entire microarray in one composite image by collecting a predefined raster of matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrometry spectra over the surface of the chip. Examining the bacterial tyrosine phosphatase YopH, we used IMS as a label-free means to visualize enzyme binding and activity with a microarrayed phosphopeptide library printed on chips coated with either gold or indium–tin oxide. Furthermore, we demonstrate that microarray-based IMS can be coupled with surface plasmon resonance imaging to add kinetic analyses to measured binding interactions. The method described here is within the capabilities of many modern MALDI–TOF instruments and has general utility for the label-free analysis of microarray assays.