Combining Pulsed SILAC Labeling and Click-Chemistry for Quantitative Secretome Analysis

Abstract

Secreted proteins, such as cytokines, chemokines, and hormones, exhibit central functions in intercellular communication, which is crucial to maintain homeostasis in every multicellular organism. A common approach to identify secreted proteins is by proteomic analysis of culture media after conditioning with a cell type of interest. This is preferably done in serum-free conditions to enable the detection of low-abundance secretory factors that would otherwise be masked by serum proteins. However, serum starvation introduces the risk of bringing cells in a stressed or perturbed state. A superior approach employs the enrichment of newly synthesized and secreted proteins from serum-containing growth medium. This is achieved by the combination of two metabolic labels: stable isotope-labeled amino acids for reliable quantification, and azidohomoalanine (AHA), an azide-bearing analogue of methionine, for the enrichment of newly synthesized and secreted proteins. This approach has been used to compare secretomes of multiple cell lines or to analyze proteins that are secreted upon a specific stimulation. Here we describe in detail the enrichment and quantification of newly synthesized and secreted proteins.