Directional cell migration requires the formation of a dominant pseudopodium in 
the direction toward which the cell migrates. When a migratory cell is 
stimulated with a chemoattractant or extracellular matrix (ECM) gradient, it 
responds with localized amplification of signals on the side facing the 
gradient. The signals mediate reorganization of the actin-myosin cytoskeleton, 
leading to morphological polarization of the cell and pseudopodium extension. To 
identify these signals, we developed an approach to biochemically isolate the 
pseudopodium from the cell body using 3.0-micrometer porous filters for 
large-scale quantitative proteomic and phosphoproteomic analysis. Here, we 
detail the methodology for pseudopodium purification and proteomic analysis. 
This model system should be widely applicable for the analysis of the 
pseudopodium proteome from various migratory cell lines, including primary and 
cancer cell lines stimulated with a diverse array of chemoattractants, ECM 
proteins, or both.