Hoorn Ewout J, Hoffert Jason D, Knepper Mark A
National Institutes of Health, 10 Center Drive, Building 10, Room 6N260, Bethesda, MD 20892, USA.
J Am Soc Nephrol. 2005 Oct;16(10):2852-63. doi: 10.1681/ASN.2005030322. Epub 2005 Aug 3.
Low sensitivity is characteristic of many proteomics methods. Presented here is an approach that combines proteomics based on difference gel electrophoresis (DIGE) with bioinformatic pathways analysis to identify both abundant and relatively nonabundant proteins in inner medullary collecting duct (IMCD) altered in abundance during escape from vasopressin-induced antidiuresis. Rats received the vasopressin analog dDAVP by osmotic minipump plus either a daily water load (vasopressin escape) or only enough water to replace losses (control). Immunoblotting confirmed the hallmark of vasopressin escape, a decrease in aquaporin-2, and demonstrated a decrease in the abundance of the urea transporter UT-A3. DIGE identified 22 mostly high-abundance proteins regulated during vasopressin escape. These proteins were analyzed using pathways analysis software to reveal protein clusters incorporating the proteins identified by DIGE. A single dominant cluster emerged that included many relatively low-abundance proteins (abundances too low for DIGE identification), including several transcription factors. Immunoblotting confirmed a decrease in total and phosphorylated c-myc, a decrease in c-fos, and increases in c-jun and p53. Furthermore, immunoblotting confirmed hypothesized changes in other proteins in the proposed network: Increases in c-src, receptor for activated C kinase 1, calreticulin, and caspase 3 and decreases in steroid receptor co-activator 1, Grp78/BiP, and annexin A4. This combined approach proved capable of uncovering regulatory proteins that are altered in response to a specific physiologic perturbation without being detected directly by DIGE. The results demonstrate a dominant protein regulatory network in IMCD cells that is altered in association with vasopressin escape, providing a new framework for further studies of signaling in IMCD.
低灵敏度是许多蛋白质组学方法的特点。本文介绍了一种将基于差异凝胶电泳（DIGE）的蛋白质组学与生物信息通路分析相结合的方法，以鉴定在从血管加压素诱导的抗利尿状态中逃脱时内髓集合管（IMCD）中丰度发生改变的丰富和相对不丰富的蛋白质。大鼠通过渗透微型泵接受血管加压素类似物dDAVP，同时给予每日水负荷（血管加压素逃脱组）或仅给予足够的水以补充损失（对照组）。免疫印迹证实了血管加压素逃脱的标志，即水通道蛋白-2减少，并表明尿素转运体UT-A3的丰度降低。DIGE鉴定出22种在血管加压素逃脱过程中受调节的主要为高丰度的蛋白质。使用通路分析软件对这些蛋白质进行分析，以揭示包含DIGE鉴定出的蛋白质的蛋白质簇。出现了一个单一的主要簇，其中包括许多相对低丰度的蛋白质（丰度太低无法通过DIGE鉴定），包括几种转录因子。免疫印迹证实总c-myc和磷酸化c-myc减少，c-fos减少，c-jun和p53增加。此外，免疫印迹证实了所提出网络中其他蛋白质的假设变化：c-src、活化C激酶1受体、钙网蛋白和半胱天冬酶3增加，类固醇受体辅激活因子1、Grp78/BiP和膜联蛋白A4减少。这种联合方法证明能够揭示因特定生理扰动而改变但未被DIGE直接检测到的调节蛋白。结果表明IMCD细胞中存在一个主要的蛋白质调节网络，该网络与血管加压素逃脱相关而发生改变，为进一步研究IMCD中的信号传导提供了一个新框架。
