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/肽（分泌组）是一个挑战。我们的实验室已经开发了一种新的技术来解决这一挑战，利用毛细管超滤（CUF）探针捕获体内分泌体。毛细管超滤涉及施加于半透膜纤维的真空以从间隙空间提取含有分泌分子的流体。CUF探针还提供了一种从活生物系统中获得小体积样品的简单方法。我们成功地通过质谱直接鉴定和测序了分泌组，绕过了主要基于二维电泳凝胶的传统蛋白质组学方法。在本研究中，我们将利用已建立的紫外线诱导的纤维肉瘤肿瘤进展/消退模型系统，利用CUF探针技术收集体内分泌物。将使用质谱法（MALDI-TOF MS和Q-TOF 2 MS/MS）和酶联免疫吸附试验（ELISA）鉴定肿瘤分泌体。同时，我们将通过高通量质谱（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.