Analysis of the cellular secretome by targeted subcellular proteomics

点击翻译按钮获取中文摘要

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.