Cell migration requires actin/myosin-mediated membrane protrusion of a 
pseudopodium (or lamellipodium) and its attachment to the substratum. This 
process guides the direction of cell movement through cytoskeletal remodeling 
and is regulated by complex signaling networks that act spatially downstream of 
integrin adhesion receptors. Understanding how these regulatory networks are 
organized in migratory cells is important for many physiological and 
pathological processes, including wound healing, immune function, and cancer 
metastasis. Here, we describe methods for the immunoaffinity purification of 
phosphotyrosine proteins (pY) from pseudopodia that have been isolated from 
migratory cells. These methods are compatible with current mass 
spectrometry-based protein identification technologies and can be utilized for 
the large-scale identification of the pseudopodium pY proteome in various 
migratory cell lines, including primary and cancer cells.