Analysis of the cellular secretome by targeted subcellular proteomics

描述（申请人提供）：分泌蛋白在细胞间通讯和控制一些生理过程中起重要作用。因此，鉴定细胞分泌的蛋白质具有重要的意义。常规的分泌组学方法通常依赖于细胞培养上清的质谱（MS）分析。然而，细胞培养液中低丰度的分泌蛋白（信号）和高浓度的血清蛋白（噪声）给细胞培养带来了重大挑战。无血清培养基的使用缓解了低信噪比问题，但反过来又会影响细胞生理。此外，细胞培养系统不能有效地再现细胞在体内的微环境。在这里，我们提出了一种替代策略的分泌组分析，以克服这些问题-靶向质谱分析富集分泌囊泡的亚细胞组分。分泌的蛋白质通过分泌囊泡运输到细胞外部。我们建议建立一种免疫沉淀法分离富含分泌囊泡的亚细胞组分；随后，这些样品的质谱分析将用于鉴定被困在分泌囊中的分泌货物。因此，在Aim 1中，我们将生成结合蛋白，该结合蛋白可用于通过类似免疫沉淀的程序从细胞裂解物中分离分泌囊泡。在Aim 2中，我们将进行质谱分析来鉴定分离的结合物的蛋白靶点

DESCRIPTION (provided by applicant): Secreted proteins play an important role in intercellular communication and control several physiological processes. Therefore identification of proteins secreted by a cell is of significant interest and importance. Conventional approaches to secretomics typically rely on mass spectrometry (MS) analysis of cell culture supernatants. However the low abundance of secreted proteins (signal) and high concentration of serum proteins (noise) in cell culture medium poses a significant challenge. The use of serum-free medium alleviates the low signal-to-noise problem but in turn can affect cellular physiology. Moreover, cell culture systems cannot effectively reproduce the microenvironment of cells in vivo. Here we propose an alternative strategy for secretome analysis to overcome these problems - targeted MS analysis of subcellular fractions enriched in secretory vesicles. Secreted proteins are transported to the cell exterior by secretory vesicles. We propose to develop an immunoprecipitation method for isolation of subcellular fractions enriched in secretory vesicles; subsequently, MS analysis of these samples will be used for identification of secretory cargo trapped in the secretory vesicles. Accordingly, in Aim 1, we will generate binding proteins that can be used to isolate secretory vesicles from cell lysates by an immunoprecipitation-like procedure. In Aim 2, we will conduct MS analysis to identify protein targets of binders isolated in Aim 1. We will also conduct MS analysis on subcellular fractions, isolated from hESCs using these binding proteins; we expect to rigorously validate that this approach can indeed be used for analysis of the cellular secretome. Finally, in Aim 3, we will investigate if our approach can be used for secretomic analysis of frozen tissues obtained from an animal cancer model. We expect that findings from our experiments and reagents developed in this project will have broad applicability, particularly in stem cell biology and tumor biomarker discovery. Our strategy can uniquely enable secretome analysis in at least three scenarios where traditional MS approaches cannot be employed: (i) to study the temporal protein secretion response to an extracellular cue, e.g. a differentiation trigger in stem cells, (ii) to identify proteins secreted by cells in a heterogeneous population provided the target cells can be isolated, e.g. using immunoaffinity methods, and (iii) secretome analysis from primary tissues such as those obtained from tumor biopsies. Notably, compatibility of this approach with frozen tissue samples will enable the use of existing human tissue repositories for discovery of secreted protein biomarkers of disease. This in turn will allow the development of minimally invasive assays for disease diagnosis.