In-depth Analyses of Kinase-dependent Tyrosine Phosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers
In-depth Analyses of Kinase-dependent Tyrosine Phosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers
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DOI：
10.1074/mcp.m110.000091
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发表时间：
2010-10-01
影响因子：
7
通讯作者：
Tao, W. Andy
中科院分区：
文献类型：
--
作者：
Iliuk, Anton B.;Martin, Victoria A.;Tao, W. Andy
深入了解细胞中信号网络的能力是系统生物学研究的一个关键目标。这种能力在很大程度上取决于磷酸化蛋白质组的无偏和可重复的分析。在这里，我们提出了一种新的蛋白质组学工具，基于聚合物的金属离子亲和捕获（PolyMAC），磷酸化肽的高效分离，以促进全面的磷酸化蛋白质组分析。这种方法使用多官能化的聚酰胺胺树枝状大分子与钛离子和醛基，使螯合和随后的分离磷酸肽在一个均匀的环境。与目前基于固相微米和纳米颗粒的策略相比，PolyMAC表现出出色的重现性，卓越的选择性，快速螯合时间，以及从复杂混合物中回收高磷酸肽。使用PolyMAC方法与抗体富集相结合，我们在恶性乳腺癌细胞中鉴定了794个独特的酪氨酸磷酸化位点，其中514个位点依赖于Syk的表达，Syk是一种具有异常肿瘤抑制因子特性的蛋白质酪氨酸激酶。PolyMAC的上级灵敏度使我们能够识别各种主要信号网络中的新组分，包括细胞迁移和凋亡。PolyMAC为磷酸化蛋白质组学和分子信号传导提供了一个强大且广泛适用的工具。Molecular & Cellular Proteomics 9：2162-2172，2010.
The ability to obtain in-depth understanding of signaling networks in cells is a key objective of systems biology research. Such ability depends largely on unbiased and reproducible analysis of phosphoproteomes. We present here a novel proteomics tool, polymer-based metal ion affinity capture (PolyMAC), for the highly efficient isolation of phosphopeptides to facilitate comprehensive phosphoproteome analyses. This approach uses polyamidoamine dendrimers multifunctionalized with titanium ions and aldehyde groups to allow the chelation and subsequent isolation of phosphopeptides in a homogeneous environment. Compared with current strategies based on solid phase micro-and nanoparticles, PolyMAC demonstrated outstanding reproducibility, exceptional selectivity, fast chelation times, and high phosphopeptide recovery from complex mixtures. Using the PolyMAC method combined with antibody enrichment, we identified 794 unique sites of tyrosine phosphorylation in malignant breast cancer cells, 514 of which are dependent on the expression of Syk, a protein-tyrosine kinase with unusual properties of a tumor suppressor. The superior sensitivity of PolyMAC allowed us to identify novel components in a variety of major signaling networks, including cell migration and apoptosis. PolyMAC offers a powerful and widely applicable tool for phosphoproteomics and molecular signaling. Molecular & Cellular Proteomics 9:2162-2172, 2010.