Analysis of secreted proteins

Abstract

Most biological processes including growth, proliferation, differentiation, and apoptosis are coordinated by tightly regulated signaling pathways, which also involve secreted proteins acting in an autocrine and/ or paracrine manner. In addition, extracellular signaling molecules affect local niche biology and in fl uence the cross-talking with the surrounding tissues. The understanding of this molecular language may provide an integrated and broader view of cellular regulatory networks under physiological and pathological conditions. In this context, the pro fi ling at a global level of cell secretomes (i.e., the subpopulations of a proteome secreted by the cell) has become an active area of research. The current interest in secretome research also deals with its high potential for the biomarker discovery and the identi fi cation of new targets for therapeutic strategies. Several proteomic and mass spectrometry platforms and methodologies have been applied to secretome pro fi ling of conditioned media of cultured cell lines and primary cells. Nevertheless, the analysis of secreted proteins is still a very challenging task, because of the technical dif fi culties that may hamper the subsequent mass spectrometry analysis. This chapter describes a typical work fl ow for the analysis of proteins secreted by cultured cells. Crucial issues related to cell culture conditions for the collection of conditioned media, secretome preparation, and mass spectrometry analysis are discussed. Furthermore, an overview of quantitative LC-MS-based approaches, computational tools for data analysis, and strategies for validation of potential secretome biomarkers is also presented.