Salisbury Cleo M, Maly Dustin J, Ellman Jonathan A
Center for New Directions in Organic Synthesis, Department of Chemistry, University of California, Berkeley, CA 94720, USA.
J Am Chem Soc. 2002 Dec 18;124(50):14868-70. doi: 10.1021/ja027477q.
A method is described for the preparation of substrate microarrays that allow for the rapid determination of protease substrate specificity. Peptidyl coumarin substrates, synthesized on solid support using standard techniques, are printed onto glass slides using DNA microarraying equipment. The linkage from the peptide to the slide is formed through a chemoselective reaction, resulting in an array of uniformly displayed fluorogenic substrates. The arrays can be treated with proteases to yield substrate specificity profiles. Standard instrumentation for visualization of microarrays can be used to obtain comparisons of the specificity constants for all of the prepared substrates. The utility of these arrays is demonstrated by the selective cleavage of preferred substrates with trypsin, thrombin, and granzyme B, and by assessing the extended substrate specificity of thrombin using a microarray of 361 different peptidyl coumarin substrates.
本文描述了一种制备底物微阵列的方法，该方法可快速测定蛋白酶底物特异性。使用标准技术在固相载体上合成的肽基香豆素底物，通过DNA微阵列设备打印到载玻片上。肽与玻片之间的连接通过化学选择性反应形成，从而得到一系列均匀展示的荧光底物阵列。这些阵列可用蛋白酶处理以产生底物特异性图谱。用于微阵列可视化的标准仪器可用于比较所有制备底物的特异性常数。通过用胰蛋白酶、凝血酶和颗粒酶B选择性切割优选底物，以及使用包含361种不同肽基香豆素底物的微阵列评估凝血酶的扩展底物特异性，证明了这些阵列的实用性。
