Protein Profiling of Active Cysteine Cathepsins in Living Cells Using an Activity-Based Probe Containing a Cell-Penetrating Peptide
Protein Profiling of Active Cysteine Cathepsins in Living Cells Using an Activity-Based Probe Containing a Cell-Penetrating Peptide
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使用含有细胞穿透肽的基于活性的探针对活细胞中的活性半胱氨酸组织蛋白酶进行蛋白质分析。
DOI：
10.1021/pr300575u
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发表时间：
2012-12-01
影响因子：
4.4
通讯作者：
Zhang, Yu-Hui
中科院分区：
文献类型：
--
作者：
Fan, Fengkai;Nie, Si;Zhang, Yu-Hui
细胞可渗透的活性探针（ABP）能够标记活细胞中的靶蛋白，从而为分析天然环境中的活性酶提供了强有力的工具。在这项研究中，我们描述了一种新的三功能细胞可渗透的活性为基础的探针（TCpABP）的蛋白质组学分析活性半胱氨酸组织蛋白酶在活细胞中的合成和使用。我们证明，虽然TCpABP含有细胞不可渗透的标签，它能够有效地进入活细胞通过传递的细胞穿透肽。TCpABP还允许同时检测和亲和分离标记的蛋白质与荧光团和生物素基序，分别。我们优化了富集方案，以最大限度地减少污染物，并确定了7个组织蛋白酶，其中2个从未被确定使用现有的ABP。我们还使用了无标记的定量方法来定量活性组织蛋白酶的相对丰度，并将其与先前公布的mRNA表达水平进行比较。除组织蛋白酶H外，大多数组织蛋白酶的mRNA表达水平和蛋白质相对活性之间存在高度相关性。本文的结果表明，TCpABP是有价值的活细胞中的活性组织蛋白酶的检测，并提供了有用的指导方针，设计新的细胞可渗透的ABP在体内标记及其在体内蛋白质组学研究中的应用。
Cell-permeable activity-based probes (ABPs) are capable of labeling target proteins in living cells, thereby providing a powerful tool for profiling active enzymes in their native environment. In this study, we describe the synthesis and use of a novel trifunctional cell-permeable activity-based probe (TCpABP) for proteomic profiling of active cysteine cathepsins in living cells. We demonstrate that although TCpABP contains cell-impermeable tags, it was able to enter living cells efficiently via the delivery of a cell-penetrating peptide. TCpABP also allowed simultaneous detection and affinity isolation of labeled proteins with a fluorophore and a biotin motif, respectively. We optimized the enrichment protocol to minimize contaminants and identified 7 cathepsins, 2 of which have never been identified using existing ABPs. We also used a label-free quantification approach to quantify the relative abundances of active cathepsins and compared them with their previously published mRNA expression levels. A high degree of correlation between the mRNA expression levels and protein relative activities was observed for most of the identified cathepsins except cathepsin H. The results herein indicate that TCpABP is valuable for the detection of active cathepsins in living cells and provides useful guidelines for designing novel cell-permeable ABPs for in vivo labeling and their applications in in vivo proteomics studies.