Secretomes captured in vivo via ultrafiltration probes

描述(由申请人提供)： 从动物体内获得低丰度和/或分泌性蛋白质/多肽(分泌体)是一个挑战。我们实验室开发了一种新的技术来应对这一挑战，它利用毛细管超滤(CUF)探针在体内捕获分泌体。毛细管超滤指的是对半透膜纤维施加真空，从间质间隙中提取含有分泌分子的液体。CUF探针还提供了一种从活的生物系统中获取小体积样本的简单方法。我们已经成功地通过质谱学直接鉴定和测序了分泌体，绕过了传统的蛋白质组学方法，这些方法主要是基于2D电泳胶。在这个方案中，我们将使用成熟的紫外线诱导的纤维肉瘤肿瘤进展者/退化者模型系统来利用CUF探针技术来收集体内的分泌物。肿瘤分泌物将使用质谱仪(MALDI-TOF MS和Q-TOF 2 MS/MS)和酶联免疫吸附试验(ELISA)进行鉴定。同时，我们将通过执行高通量质谱学(MALDI-TOF-TOF)和细胞因子抗体阵列来优化这项技术。我们相信，使用CUF探针/质谱仪将是一个强大的工具，可以应用于其他动物模型和其他人类疾病。

DESCRIPTION (provided by applicant): It is a challenge to obtain low abundant and/or secretory proteins/peptides (secretomes) from animals in vivo. Our laboratory has developed a novel technique to address this challenge which utilizes capillary ultrafiltration (CUF) probes to capture secretomes in vivo. Capillary ultrafiltration involves a vacuum applied to semipermeable membrane fibers to extract fluid containing secretory molecules from the interstitial space. The CUF probes also provide a simple means to obtain a small-volume sample from living biological systems. We have successfully identified and sequenced the secretomes directly by mass spectrometry, bypassing the traditional proteomics approaches which are primarily based on the 2D electrophoresis gels. In this proposal, we will use the well-established UV-induced fibrosarcoma tumor progressor/regressor model system to exploit the technique of CUF probes to collect in vivo secretomes. Tumor secretomes will be identified using mass spectrometry (MALDI-TOF MS and Q-TOF 2 MS/MS) and enzyme-linked immunosorbent assay (ELISA). In parallel, we will optimize this technique by performing high-throughput mass spectrometry (MALDI-TOF-TOF) and Cytokine Antibody Arrays. We believe that using CUF probes/Mass spectrometry will be a powerful tool which can be applied to other animal models and other human diseases.