Developing photoactive affinity probes for proteomic profiling: Hydroxamate-based probes for metalloproteases
Developing photoactive affinity probes for proteomic profiling: Hydroxamate-based probes for metalloproteases
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DOI：
10.1021/ja047044i
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发表时间：
2004-11-10
影响因子：
15
通讯作者：
Yao, SQ
中科院分区：
文献类型：
--
作者：
Chan, EWS;Chattopadhaya, S;Yao, SQ
目前基于活性的蛋白质分析策略的变性方面限制了化学探针的类别，这些探针可以不可逆地和共价地修饰它们的靶酶。在这里，我们提出了一种免费的，基于亲和的标记方法来分析不具有共价结合的底物中间体的酶。使用多种属于金属蛋白酶类的酶，该方法的可行性在几个概念验证实验中得到了成功证明。以金属蛋白酶为目标的亲和探针的设计模板由一个肽基羟酸锌结合基（ZBG）、一个荧光报告标记和一个光耐受重氮嘧啶基组成。探针中光降解单元的光解有效地在探针和目标酶之间产生共价的，不可逆的连接，使酶在SDS-PAGE凝胶上分离时与未标记的蛋白质区分开来。进行了各种标记研究，以证实基于亲和的方法选择性地标记金属蛋白酶存在大量过量的其他蛋白质，并且标记反应的成功密切依赖于酶的催化活性。竞争性抑制剂的添加按比例减少了酶标记的程度，使该方法对金属蛋白酶抑制剂的潜在原位筛选有用。使用不同的探针与不同的P，氨基酸，我们能够产生独特的“指纹”谱的酶，可用于确定其底物特异性。最后，通过对一组酵母金属蛋白酶的测试，我们证明了基于亲和的方法可以在未来的蛋白质组学实验中用于金属蛋白酶的大规模分析。
The denaturing aspect of current activity-based protein profiling strategies limits the classes of chemical probes to those which irreversibly and covalently modify their targeting enzymes. Herein, we present a complimentary, affinity-based labeling approach to profile enzymes which do not possess covalently bound substrate intermediates. Using a variety of enzymes belonging to the class of metalloproteases, the feasibility of the approach was successfully demonstrated in several proof-of-concept experiments. The design template of affinity-based probes targeting metalloproteases consists of a peptidyl hydroxamate zinc-binding group (ZBG), a fluorescent reporter tag, and a photolabile diazirine group. Photolysis of the photolabile unit in the probe effectively generates a covalent, irreversible linkage between the probe and the target enzyme, rendering the enzyme distinguishable from unlabeled proteins upon separation on a SDS-PAGE gel. A variety of labeling studies were carried out to confirm that the affinity-based approach selectively labeled metalloproteases in the presence of a large excess of other proteins and that the success of the labeling reaction depends intimately upon the catalytic activity of the enzyme. Addition of competitive inhibitors proportionally diminished the extent of enzyme labeling, making the approach useful for potential in situ screening of metalloprotease inhibitors. Using different probes with varying P, amino acids, we were able to generate unique "fingerprint" profiles of enzymes which may be used to determine their substrate specificities. Finally, by testing against a panel of yeast metalloproteases, we demonstrated that the affinity-based approach may be used for the large-scale profiling of metalloproteases in future proteomic experiments.