Proteomics evaluation of chemically cleavable activity-based probes
Proteomics evaluation of chemically cleavable activity-based probes
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DOI：
10.1074/mcp.m700124-mcp200
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发表时间：
2007-10-01
影响因子：
7
通讯作者：
Bogyo, Matthew
中科院分区：
文献类型：
--
作者：
Fonovic, Marko;Verhelst, Steven H. L.;Bogyo, Matthew
特异性靶向相关酶蛋白亚群的基于活性的探针（ABP）在蛋白质组学研究中的应用越来越多。这些试剂的主要应用之一是探针标记靶的亲和分离。然而，在ABP上使用廉价且有效的生物素亲和标签可能是有问题的，因为难以释放捕获的蛋白质。在这里，我们描述了基于活性的探针进行化学可裂解的接头，允许选择性释放的探针标记的蛋白质在温和的洗脱条件下，与质谱分析兼容的评价。具体而言，我们比较了亲和纯化后探针标记靶标的标准珠上消化结果与使用化学选择性切割获得的结果。结果显示了靶向丝氨酸和半胱氨酸蛋白酶的多种APB。这些结果突出了通过使用可裂解接头系统获得的数据质量的显著改善。
Activity-based probes (ABPs) that specifically target subsets of related enzymatic proteins are finding increasing use in proteomics research. One of the main applications for these reagents is affinity isolation of probe-labeled targets. However, the use of cheap and efficient biotin affinity tags on ABPs can be problematic due to difficulty in release of captured proteins. Here we describe the evaluation of activity-based probes carrying a chemically cleavable linker that allows selective release of probe-labeled proteins under mild elution conditions that are compatible with mass spectrometric analysis. Specifically, we compare results from standard on-bead digestion of probe-labeled targets after affinity purification with the results obtained using chemoselective cleavage. Results are presented for multiple APBs that target both serine and cysteine proteases. These results highlight significant improvements in the quality of data obtained by using the cleavable linker system.