Identification of proteins that associate with integrin α2 by proteomic analysis in human fibrosarcoma HT-1080 cells.
Identification of proteins that associate with integrin α2 by proteomic analysis in human fibrosarcoma HT-1080 cells.
复制标题
通过人纤维肉瘤 HT-1080 细胞中的蛋白质组学分析鉴定与整合素 α2 相关的蛋白质。
DOI：
10.1002/jcp.23054
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Koshikawa N.
中科院分区：
文献类型：
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作者：
Uematsu T;Konishi C;Hoshino D;HanX;Tomari T;Egawa N;Takada Y;IsobeT;Seiki M;Koshikawa N.
整合素是细胞外基质 (ECM) 成分的粘附受体，通过介导双向信号传输来调节多种细胞功能，例如迁移、侵袭、增殖和存活。尽管据报道许多蛋白质与细胞表面和细胞内的整合素相关，但尚未对粘附体进行系统分析。在之前的研究中，我们使用纳流液相色谱/串联质谱（nano-LC/MS/MS）对在优化细胞裂解和纯化条件下制备的相关蛋白进行鉴定，从而鉴定了与膜型蛋白酶 MT1-MMP 相关的蛋白。由于整合素被鉴定为 MT1-MMP 相关蛋白，我们接下来应用该方法来分析整合素相关蛋白。在这项研究中，我们在 HT1080 细胞中表达了 C 末端与 FLAG 肽融合的整合素 α2。用 1% Brij-98 裂解稳定表达嵌合蛋白的细胞，并使用抗 FLAG 抗体进行亲和纯化。整合素 β1 与整合素 α2 共纯化，证实了纯化过程的特异性。通过 Nano-LC/MS/MS 对纯化混合物进行分析，鉴定出 70 种蛋白质。其中十九种是膜蛋白，包括粘附蛋白、受体、转运蛋白、蛋白酶和离子通道受体，其余的是细胞质。有趣的是，其中八种蛋白质之前已被证明与 MT1-MMP 相关。我们相信本研究为促进细胞粘附、迁移和侵袭机制的研究提供了一个平台。 J.细胞。生理学。 227：3072–3079，2012 年。© 2011 Wiley 期刊公司。
Integrins are adhesion receptors for components of the extracellular matrix (ECMs) that regulate multiple cellular functions, such as migration, invasion, proliferation, and survival by mediating bidirectional signal transmission. Even though many proteins have been reported to associate with integrins both on and in cells, systemic analyses of the adhesome have not been carried out. In previous studies, we identified proteins associating with a membrane‐type protease, MT1‐MMP, using nano‐flow liquid chromatography/tandem mass spectrometry (nano‐LC/MS/MS) of associated proteins prepared by optimized conditions for cell lysis and purification. Since integrins were identified as MT1‐MMP‐associated proteins, we next applied this method to analyze integrin‐associated proteins. In this study, we expressed integrin α2 fused at the C terminus to a FLAG peptide in HT1080 cells. Cells stably expressing the chimeric protein were lysed with 1% Brij‐98 and affinity purified using anti‐FLAG antibody. Integrin β1 co‐purified with integrin α2 confirming the specificity of the purification procedure. Analysis of the purified mixture by nano‐LC/MS/MS identified 70 proteins. Nineteen of these were membrane proteins, including adhesion proteins, receptors, transporters, proteinases, and ion‐channel receptors, and the balance were cytoplasmic. Interestingly, eight of the proteins had previously been shown to associate with MT1‐MMP. We believe the present study provides a platform to facilitate the study of the mechanisms of cell adhesion, migration, and invasion. J. Cell. Physiol. 227: 3072–3079, 2012. © 2011 Wiley Periodicals, Inc.