A Proteomic Protocol to Identify Physiological Substrates of Pro-protein Convertases

Proprotein convertases (PCs) convert pro-proteins into their bioactive forms through limited proteolytic cleavage, thereby regulating the temporal and spatial activation of a large number of functionally important proteins. This “converting” process is involved in a wide range of essential physiological and pathological processes, making PCs valuable therapeutic targets. One of the challenges in the field of PC research has been to identify the physiological substrates of a particular PC in a specific tissue or cellular process. Proteomics provides an unprecedented opportunity to identify novel PC substrates in a physiological context. Here we provide a detailed practical procedure utilizing two-dimensional fluorescent differential gel electrophoresis (2D-DiGE) and tandem mass spectrometry techniques, in combination with other standard molecular and biochemical methods, to identify and subsequently validate novel PC6 substrates in a critical uterine event called decidualization. This method is applicable to the study of any PC members and their relevant cellular processes.